Bi-functional plasmid that can act as both a DNA vaccine and a recombinant virus vector

ABSTRACT

Polyenv vaccines are provided that comprise mixtures of at least 4 to about 10,000 different recombinant viruses that each express a different HIV env variant or a portion thereof containing both constant and variable regions, as well as methods of making and using such polyenv vaccines and viruses, including the use of the polyenv vaccine, in live, attenuated or inactivated form, for prophylaxis or treatment of HIV infection. The viral vaccines of the invention are optimally combined with a recombinant HIV env booster, or a recombinant HIV env gene DNA priming or boosting vaccine.

This work was supported in part by NCI grants R01-CA57419-03 and CancerCenter Support Core Grant P30-CA21765, NIH-NIAID grants AI-32529 andP01-AI31596-04. Accordingly, the U.S. Government has certain rights inthis invention.

CONTINUING INFORMATION

The present Application is a Division of application Ser. No.08/788,815, filed Jan. 23, 1997, now U.S. Pat. No. 5,846,546, issuedDec. 8, 1998 which is a Continuation-In-Part of application Ser. No.08/590,288, filed Jan. 23, 1996, now U.S. Pat. No. 5,741,492, issuedApr. 21, 1998, the disclosures of which are incorporated herein byreference in their entireties. Applicants claim the benefits of theseApplications under 35 U.S.C. §120.

FIELD OF THE INVENTION

The present invention relates to polyenv vaccines for humanimmunodeficiency virus (HIV), comprising a mixture of at least 4-40 andup to 10,000 recombinant vaccinia viruses that each express a differentvariant of an HIV envelope protein. The vaccines are suitable for thevaccination of mammals, including humans, in order to provideunexpectedly enhanced cellular and/or humoral immune responses to HIVinfection. Additionally, the invention relates to methods for making andusing such recombinant vaccinia viruses and polyenv vaccines.

BACKGROUND OF THE INVENTION

The AIDS virus is likely to claim tens of millions of lives by the year2,000, constituting a worldwide health concern of top priority [see,DeVita, et al., AIDS, Etiology, Diagnosis, Treatment and Prevention, 3rdedition, J. B. Lippincott Co., Philadelphia, Pa. (1992); Wong-Staal, inVirology, pp 1529-1543; and Hirsch, et al., in Virology, pp. 1545-1570].The design of an effective HIV vaccine poses a particular challenge toimmunologists, as the reverse transcriptase enzyme involved in thereplication of HIV has a high error rate. This results in many mutantHIV strains having outer coat or envelope proteins with variant proteinsequences. These variant envelope proteins are often recognized asdifferent antigens by the mammalian immune system, which produces morethan 10⁹ new lymphocytes per day for the sole purpose of counteringforeign antigens. B and T-cells constitute, respectively, the humoraland cellular components of the immune response.

A good example of the qualitative strength of such immune responses isshown in HIV-infected patients and in SIV-infected macaques. In eachcase, successive rounds of infection, immunity, and establishment ofvariant HIVs or SIVs occur [Wrin, et al., J. Acquir. Immune Defic.Syndr. 7:211-219 (1994); Burns and Desrosiers, Cur. Topics Microbiol.Immunol. 188:185-219 (1994)]. With each cycle, the diversity of HIVantigenic determinants (and the corresponding immune responses) areincreased, such that these immune responses neutralize a broad range ofSIV or HIV variants, and superinfection is largely inhibited.

However, AIDS patients develop compromised immune responses that becomeinsufficient to prevent the HIV viral infection from overcoming thepatient's immune system. This may be due in part to the establishment ofHIV variants whose envelope variant proteins are not recognized by thepatient's immune system and thus escape destruction (Sci. Amer. August1995, pp ). In such cases, even if the immune response is capable ofpreventing de novo infection (e.g., persistent mutation of the virus inprivileged sequestered sites), the HIV infection may ultimately overcomethe patient's immune response [Pantaleo et al., Nature 362:355-358(1993); Embretson, et al., Nature 362:359-362 (1993)].

The identification of B- and T-cell antigenic determinants among HIVproteins remains incomplete. The HIV envelope protein has beencharacterized as having variable (V1-V5) and constant (C1-C5) regions. Apeptide representative of the V3 region has been termed the principalneutralizing determinant (PND) [Javaherian et al., Proc. Natl. Acad.Sci. (USA) 86:6768-6772 (1989)], although other regions of the envelopeprotein may also be involved in eliciting an immune response. The fulllength envelope protein from HIV contains about 850 to 900 amino acids,with the variation in length due to hypermutation [Starcich et al., Cell45:637 (1986)].

The first vaccines against HIV evaluated in clinical trials weredesigned to present single envelope proteins, or portions thereof, tothe immune system. However, neutralizing responses towards a single or afew envelope proteins did not recognize diverse isolates of HIV and theindividuals were not protected from infection [Belshe et al., J. Am.Med. Assoc. 272:431-431 (1994); U.S. Pat. No. 5,169,763; PCT publicationWO 87/06262; Zagury et al., Nature 332:728-731 (1988); Kieny et al.,Int. Conf. AIDS 5:541 (1989); Eichberg, Int. Conf. AIDS 7:88 (1991);Cooney et al., Proc. Natl. Acad. Sci. USA 90:1882-1886 (1993), Graham etal., J. Infect. Dis. 166:244-252 (1992); J. Infect. Dis. 167:533-537(1993); Keefer et al., AIDS Res. Hum. Retrovir. 10 (Suppl. 2):S139-143(1994); Gorse, AIDS Res. Hum. Retrovir. 10 (Suppl. 2):141-143 (1994);McElrath et al., J. Infect. Dis. 169:41-47 (1994); Fauci, Science264:1072-1073 (May 1994)].

Accordingly, there is a long-felt and pressing need to discover vaccinesand methods that elicit an immune response that is sufficient to treator prevent HIV infections.

SUMMARY OF THE INVENTION

The present invention is intended to overcome one or more deficienciesof the related arts. In particular, the polyenv vaccine of the inventionadvantageously provides a more robust immune response. The strength ofthe present invention lies in its power to recruit B cell, helper Tcell, and cytotoxic T cell compartments of the immune response foreffective humoral and cellular immunity. For example, the presentinvention elicits a great breadth of HIV-specific antibody activities.HIV neutralization assays demonstrate that the antibodies elicited areof superior quality. Surprisingly, the invention can generate immuneresponses against "naive" HIV strains, i.e., HIV strains for whichenvelope proteins are not included in the polyenv cocktail.

To provide more effective HIV vaccines, the present invention providespolyenv vaccines comprising mixtures of at least 4 up to about 10,000,preferably 4 to about 1,000, and more preferably about 10 to about 100,different recombinant viruses, each expressing a different HIV envelopeprotein variant (EPV) (or a substantial portion thereof) that includesboth constant and variable regions of the envelope protein. Preferably,each of the expressed envelope protein variants have a structure and/orimmunogenicity similar to that of a native HIV envelope protein existingin an infected cell or HIV lipid bilayer, such as in an oligomeric form.Also provided are methods of making and using such recombinant virusesand polyenv vaccines. In their use as a vaccine, each of the variantenvelope proteins preferably induces a different subset of B and/or Tcells, each subset responding to different envelope proteins and, hence,to multiple HIV variants. A mixture of this number, type and/orstructure of envelope proteins is a now-discovered method for elicitinga strong, durable HIV-specific immune response with broad spectrumneutralizing activity.

In a preferred embodiment, the recombinant viruses are selected from thegroup consisting of vaccinia, canary pox virus, adenovirus, andadeno-associated virus (AAV). In a specific example, infra, vacciniavirus is used to prepare a polyenv vaccine. In a preferred embodiment, arecombinant vaccinia virus vaccine of the invention is administeredsubcutaneously. A further advantage of the invention is thatsubcutaneous administration of vaccinia virus does not result information of a lesion, thus avoiding release of infectious vaccinia,which is a potential threat to an immunocompromised population.

Preferably, a recombinant virus polyenv vaccine of the inventioncomprises a lysate of the virus-infected growth cells, e.g., vero cells,which contains expressed envelope protein variants in addition toinfectious virus. Inclusion of the lysate envelope protein variants,which abets the immune response, represents a particular distinction ofthe present invention, as generally virus is purified away from thegrowth cell lysate.

In the vaccines of the invention, the EV nucleotide may be isolated frompatients infected with an HIV virus from a geographically restrictedarea, from patients infected with an HIV virus from different clades, orfrom laboratory isolates of HIV.

The present inventors have discovered that polyenv vaccines of thepresent invention elicit unexpectedly enhanced immune responses by theexpression and/or presentation of multiple envelope protein variants,each containing both constant and variable regions, preferably having astructure that is substantially similar to that of a native HIV envelopeprotein. The enhanced immune responses recognize HIV strains in additionto those strains expressing the envelope proteins provided in thepolyenv vaccine. Thus, the aim of such a vaccine is to provide enhancedimmune responses to a wide range of HIV strains, which immune responsesare suitable for treating or preventing infection (or continuedinfection due to mutation) by different strains of the virus.

The present invention also provides env variant (EV) nucleic acidencoding (or complementary to) at least one antigenic determinant of anenvelope protein variant (EPV). The EPV is preferably encoded by arecombinant virus, as further provided in a polyenv vaccine of thepresent invention. The variant nucleic acid comprises at least onemutation that confers differing antigenic properties, or threedimensional structure, to the encoded EPV.

The present invention also provides a vaccine composition comprising apolyenv vaccine of the present invention, and a pharmaceuticallyacceptable carrier or diluent. The vaccine composition can furthercomprise an adjuvant and/or cytokine which enhances a polyenv vaccineimmune response to at least one HIV strain in a mammal administered thevaccine composition. A polyenv vaccine of the present invention iscapable of inducing an immune response inclusive of at least one of ahumoral immune response (e.g., antibodies) and a cellular immuneresponse (e.g., activation of B cells, helper T cells, and cytotoxic Tcells (CTLs)).

The present invention also provides a method for eliciting an immuneresponse to an HIV infection in a mammal which is prophylactic for anHIV infection, the method comprising administering to a mammal a vaccinecomposition comprising a polyenv vaccine of the present invention, whichis protective for the mammal against a clinical HIV-related pathologycaused by infection of at least one HIV strain.

The present invention also provides a method for eliciting an immuneresponse to an HIV infection in a mammal for therapy of an HIVinfection. The method comprises administering to a mammal a compositioncomprising an inactivated or attenuated polyenv vaccine of the presentinvention, which composition elicits an enhanced immune response,relative to controls, in the mammal against a clinical virus pathologycaused by infection with at least one HIV strain.

In a further embodiment, the prophylactic or therapeutic method ofeliciting an immune response to HIV comprising administering aneffective amount of another (e.g., second) polyenv vaccine comprising atleast 4 to about 10,000 different recombinant viruses, in which therecombinant viruses are of a different species from the recombinantviruses of the preceding vaccine, and each of the recombinant viruses inthe polyenv comprises an env variant nucleotide encoding a differentenvelope protein variant of an HIV envelope protein.

The HIV-specific immune response generated with the polyenv recombinantvirus vaccine of the invention can be further augmented by priming orboosting a humoral or cellular immune response, or both, byadministering an effective amount of at least one recombinant HIV envprotein, or a DNA vaccine, or both. Preferably the recombinant proteinor DNA vaccine is also a polyenv vaccine. Any of the vaccine strategiesprovided herein can be provided in any order. For example, a subject maybe primed with a recombinant virus polyenv vaccine, followed by boostingwith a DNA vaccine, with a final boost with a recombinant proteinvaccine. Preferably, the recombinant HIV env protein is in an admixturewith an adjuvant. In a specific embodiment, exemplified infra, therecombinant HIV env protein is administered intramuscularly. Preferably,a DNA vaccine is administered with a gene gun.

The foregoing methods of the invention provide the incentive togenetically engineer a new plasmid vector. Thus, in a corollary aspect,the present invention provides a bi-functional plasmid that can serve asa DNA vaccine and a recombinant virus vector, comprising a heterologousinsertion site under control of both an animal expression controlsequence, and a viral expression control sequence. Preferably, theanimal expression control sequence is a cytomegalovirus immediate early(CMV) promoter, and the virus expression control sequence is a vacciniavirus early promoter, a vaccinia virus late promoter, or both.

Other objects, features, advantages, utilities and embodiments of thepresent invention will be apparent to skilled practitioners from thefollowing detailed description and examples relating to the presentinvention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Schematic representation of the orientation of the HIV-1 gene ina vaccinia virus genome. The HIV-1 envelope gene is positioned betweenright and left segments of the thymidine kinase locus. A HindIII siteexists at the C-terminus of the HIV-1 envelope gene. The appropriateinsertion yields a HindIII fragment of approximately 7 kb in size.Southern blots with this pattern confirmed the position and correctorientation of the HIV-1 envelope gene.

FIG. 2. Graphical representation of data showing that the HIV-specificantibody response is long term in mammal models. The results ofrepresentative mouse sera tested in the ELISA for HIV-specificantibodies are shown. Each sample was diluted 1:100 (solid bars),1:1,000 (hatched bars) and 1:10,000 (clear bars) prior to assay onHIV-1-coated ELISA plates. Test mice were sampled at various times (1month, 4 months and 6 months) following the injection of 10⁷ pfu of avaccinia virus construct expressing one envelope protein of HIV-1. Thecontrol mouse was immunized with a vaccinia virus containing no envelopesequence. Standard error bars are shown.

FIG. 3. Graphical representation of data showing how the vaccinia virusdose affects the induction of at least one immune response, includingHIV-specific antibody production. Representative mouse serum sampleswere tested by the ELISA on HIV-1-coated plates. Serum samples weretaken from mice injected with 10⁵, 10⁶, and 10⁷ pfu of one vacciniavirus expressing the HIV-1-envelope protein. Serum samples were testedapproximately three weeks after injection. Each sample was diluted 1:100(solid bars), 1:1,000 (hatched bars) and 1:10,000 (clear bars) prior toassay on HIV-1-coated ELISA plates. Standard error bars are shown.

FIG. 4. Graphical representation of data showing that the mixing ofvaccinia virus constructs does not compromise the elicitation ofHIV-specific antibody in injected mammals. Representative mouse serumsamples were tested by the ELISA approximately 2 months following theinjection of 10⁷ pfu vaccinia virus expressing HIV-1 envelopeprotein(s). "Single" identifies a sample from am mouse that received asingle vaccinia virus. "Mix" represents a sample from a mouse thatreceived a mixture of vaccinia viruses expressing five distinct envelopeproteins. Each sample was diluted 1:100 (solid bars), 1:1,000 (hatchedbars) and 1:10,000 (clear bars) prior to assay on HIV-1-coated ELISAplates. Standard error bars are shown.

FIG. 5. Production of novel vaccinia virus recombination by thesubstitution of PCR products for pEvenv4 BH10 sequences. The method ofsequence substitution is shown. PCR products were substituted forrespective BH10 env sequences at the unique enzyme restriction sites ofKpnI and BsmI. Following the cutting of plasmid and ligation with PCRproducts, new plasmids were recombined with the wildtype VV to createVV-expression vectors.

FIG. 6. Responses in the Abbott ELISA following immunization. Sera fromall four chimpanzees were tested with the Abbott clinical assay (seeMaterials and Methods, infra). Results for each serum sample (Y-axis)are recorded for each test date (X-axis). High responses were observedin chimps immunized with the mixed VVenv vaccine.

FIG. 7. Map of bi-functional plasmid that can act both as a DNA vaccineand as a VV recombination vector. The presence of cytomegalovirusimmediate early (CMV) promoter and vaccinia virus (VV) late and earlypromoters permit expression of the foreign gene in both mammalian cellsor VV infected cells.

DETAILED DESCRIPTION OF THE DISCLOSURE

Discovery of Unexpectedly Enhanced Immune Responses to Mixed HIV PolyenvVaccines. Previous attempts to provide vaccines against differentstrains of HIV have focused on one or more variable regions of gp120 orgp160. It was expected that such variable regions, provided in avaccine, would provide broad protection against HIV infection. However,such vaccines have not been successful, where the vaccine-induced immuneresponse does not recognize many different strains of HIV. Therefore, acritical need exists to provide vaccines that elicit immune responses tomultiple strains of HIV, such that the vaccines are suitable fortreatment and/or prevention of HIV.

The present inventors have discovered that unexpectedly enhanced primaryand secondary (boosting) immune responses can be induced against severalor many different HIV strains, by the use of polyenv vaccines thatcontain a mixture of at least 4, up to as many as 1,000, and possibly asmany as 10,000, recombinant viruses that each encode a differentenvelope protein variant (EPV). The vaccine can also contain EPVsexpressed by the viruses, e.g., as produced in the host cells used forvirus production.

The terms "priming" or "primary" and "boost" or "boosting" are usedherein to refer to the initial and subsequent immunizations,respectfully, i.e., in accordance with the definitions these termsnormally have in immunology.

The EPV encoding nucleic acid (envelope variant (EV) nucleic acid) canbe isolated from the same or different population (e.g., geographic) ofhumans infected with HIV. Alternatively, the different EV nucleic acidscan be obtained from any source and selected based on screening of thesequences for differences in coding sequence or by evaluatingdifferences in elicited humoral and/or cellular immune responses tomultiple HIV strains, in vitro or in vivo, according to known methods.

The initial discovery related to recombinant vaccinia virus vaccines.However, as can be readily appreciated by one of ordinary skill in theart, any recombinant virus can be used to express polyenv antigens for avaccine of the invention. Furthermore, the use of multiple viralvaccines can obviate anti-viral immune responses that may render abooster with the viral vaccine less effective (due to possiblepotentiation of a vigorous anti-virus response).

As is readily appreciated by one of skill in the art, the inventors havefurther found that boosting with recombinant HIV env protein orproteins, preferably proteins, further potentiates the immunizationmethods of the invention. The HIV env protein or proteins may correspondto the HIV env proteins expressed in the polyenv vaccine, or they may bedifferent HIV env proteins.

Similarly, as can be appreciated by the skilled artisan, theimmunization methods of the present invention are enhanced by use of aDNA vaccine. The DNA vaccine can be used as a boost, e.g., as describedabove with respect to the recombinant HIV proteins. Alternatively, theDNA vaccine can be used to prime immunity, with the recombinant viralvaccine or vaccines used to boost the anti-HIV immune response. As withthe recombinant env protein booster vaccine, the DNA vaccine maycomprise one or more vectors for expression of one or more HIV envgenes. In addition, the HIV env genes may correspond to genes expressedby the recombinant virus vaccine, or they may be different. In apreferred embodiment, vectors are prepared for expression in therecombinant virus vaccine and in transfected mammalian cells as part ofa DNA vaccine.

This immune response (as humoral and/or cellular) is found to beeffective for a broader range of strains of an infectious virus, such asHIV, and is not limited to the virus strains expressing the specificenvelope protein variants (EPVs) provided by the polyenv vaccine. Thepresent invention thus provides multiple EPVs encoded by a recombinantviral vaccine which give unexpectedly enhanced immune responses tomultiple strains of HIV.

Polyenv Vaccines and Vaccination

The present invention thus provides, in one aspect, polyenv vaccinesusing mixtures of at least 4, and up to 10,000 different recombinantvaccinia viruses that each express a different envelope protein variant,or an antigenic portion thereof. As can be readily appreciated to one ofskill in the art, 4 to about 1000, or preferably about 10 to about 100,different recombinant viruses could be employed. One of ordinary skillin the art can further readily appreciate that other viruses can be usedfor vaccines. Examples of suitable viruses that can act as recombinantviral hosts for vaccines, in addition to vaccinia, includes canarypox,adenovirus, and adeno-associated virus. Also provided are methods ofmaking and using such polyenv vaccines.

A polyenv vaccine of the present invention induces at least one of ahumoral and a cellular immune response in a mammal who has beenadministered the polyenv vaccine, but the response to the vaccine issubclinical, or is effective in enhancing at least one immune responseto at least one strain of HIV, such that the vaccine administration issuitable for vaccination purposes.

Viral vaccines. Various genetically engineered virus hosts ("recombinantviruses") can be used to prepare polyenv viral vaccines foradministration of HIV polyenv antigens. Viral vaccines are particularlyadvantageous, in that the viral infection component promotes a vigorousimmune response that targets activation of B lymphocytes, helper Tlymphocytes, and cytotoxic T lymphocytes. Numerous virus species can beused as the recombinant virus hosts for the vaccines of the invention. Apreferred recombinant virus for a viral vaccine is vaccinia virus[International Patent Publication WO 87/06262, Oct. 22, 1987, by Moss etal.; Cooney et al., Proc. Natl. Acad. Sci. USA 90:1882-6 (1993); Grahamet al., J. Infect. Dis. 166:244-52 (1992); McElrath et al., J. Infect.Dis. 169:41-7 (1994)]. In another embodiment, recombinant canarypox canbe used [Pialoux et al., AIDS Res. Hum. Retroviruses 11:373-81 (1995),erratum in AIDS Res. Hum. Retroviruses 11:875 (1995); Andersson et al.,J. Infect. Dis. 174:977-85 (1996); Fries et al., Vaccine 14:428-34(1996); Gonczol et al., Vaccine 13:1080-5 (1995)]. Another alternativeis defective adenovirus or adenovirus [Gilardi-Hebenstreit et al., J.Gen. Virol. 71:2425-31 (1990); Prevec et al., J. Infect. Dis. 161:27-30(1990); Lubeck et al., Proc. Natl. Acad. Sci. USA 86:6763-7 (1989);Xiang et al., Virology 219:220-7 (1996)]. Other suitable viral vectorsinclude retroviruses that are packaged in cells with amphotropic hostrange [see Miller, Human Gene Ther. 1:5-14 (1990); Ausubel et al.,Current Protocols in Molecular Biology, § 9], and attenuated ordefective DNA virus, such as but not limited to herpes simplex virus(HSV) [see, e.g., Kaplitt et al., Molec. Cell. Neurosci. 2:320-330(1991)], papillomavirus, Epstein Barr virus (EBV), adeno-associatedvirus (AAV) [see, e.g., Samulski et al., J. Virol. 61:3096-3101 (1987);Samulski et al., J. Virol. 63:3822-3828 (1989)], and the like.

DNA vaccines. An alternative to a traditional vaccine comprising anantigen and an adjuvant involves the direct in vivo introduction of DNAencoding the antigen into tissues of a subject for expression of theantigen by the cells of the subject's tissue. Such vaccines are termedherein "DNA vaccines" or "nucleic acid-based vaccines." DNA vaccines aredescribed in International Patent Publication WO 95/20660 andInternational Patent Publication WO 93/19183, the disclosures of whichare hereby incorporated by reference in their entireties. The ability ofdirectly injected DNA that encodes a viral protein to elicit aprotective immune response has been demonstrated in numerousexperimental systems [Conry et al., Cancer Res., 54:1164-1168 (1994);Cox et al., Virol, 67:5664-5667 (1993); Davis et al., Hum. Mole. Genet.,2:1847-1851 (1993); Sedegah et al., Proc. Natl. Acad. Sci., 91:9866-9870(1994); Montgomery et al., DNA Cell Bio., 12:777-783 (1993); Ulmer etal., Science, 259:1745-1749 (1993); Wang et al., Proc. Natl. Acad. Sci.,90:4156-4160 (1993); Xiang et al., Virology, 199:132-140 (1994)].Studies to assess this strategy in neutralization of influenza virushave used both envelope and internal viral proteins to induce theproduction of antibodies, but in particular have focused on the viralhemagglutinin protein (HA) [Fynan et al., DNA Cell. Biol., 12:785-789(1993A); Fynan et al., Proc. Natl. Acad. Sci., 90:11478-11482 (1993B);Robinson et al., Vaccine, 11:957, (1993); Webster et al., Vaccine,12:1495-1498 (1994)].

Vaccination through directly introducing DNA that encodes an HIV envprotein to elicit a protective immune response produces bothcell-mediated and humoral responses. This is analogous to resultsobtained with live viruses [Raz et al., Proc. Natl. Acad. Sci.,91:9519-9523 (1994); Ulmer, 1993, supra; Wang, 1993, supra; Xiang, 1994,supra]. Studies with ferrets indicate that DNA vaccines againstconserved internal viral proteins of influenza, together with surfaceglycoproteins, are more effective against antigenic variants ofinfluenza virus than are either inactivated or subvirion vaccines[Donnelly et al., Nat. Medicine, 6:583-587 (1995)]. Indeed, reproducibleimmune responses to DNA encoding nucleoprotein that last essentially forthe lifetime of the animal have been reported in mice [Yankauckas etal., DNA Cell Biol., 12: 771-776 (1993)].

As is well known in the art, a large number of factors can influence theefficiency of expression of antigen genes and/or the immunogenicity ofDNA vaccines. Examples of such factors include the reproducibility ofinoculation, construction of the plasmid vector, choice of the promoterused to drive antigen gene expression and stability of the inserted genein the plasmid. Depending on their origin, promoters differ in tissuespecificity and efficiency in initiating mRNA synthesis [Xiang et al.,Virology, 209:564-579 (1994); Chapman et al., Nucle. Acids. Res.,19:3979-3986 (1991)]. To date, most DNA vaccines in mammalian systemshave relied upon viral promoters derived from cytomegalovirus (CMV).These have had good efficiency in both muscle and skin inoculation in anumber of mammalian species. Another factor known to affect the immuneresponse elicited by DNA immunization is the method of DNA delivery;parenteral routes can yield low rates of gene transfer and produceconsiderable variability of gene expression [Montgomery, 1993, supra].High-velocity inoculation of plasmids, using a gene-gun, enhanced theimmune responses of mice [Fynan, 1993B, supra; Eisenbraun et al., DNACell Biol., 12: 791-797 (1993)], presumably because of a greaterefficiency of DNA transfection and more effective antigen presentationby dendritic cells. Vectors containing the nucleic acid-based vaccine ofthe invention may also be introduced into the desired host by othermethods known in the art, e.g., transfection, electroporation,microinjection, transduction, cell fusion, DEAE dextran, calciumphosphate precipitation, lipofection (lysosome fusion), or a DNA vectortransporter [see, e.g., Wu et al., J. Biol. Chem. 267:963-967 (1992); Wuand Wu, J. Biol. Chem. 263:14621-14624 (1988); Hartmut et al., CanadianPatent Application No. 2,012,311, filed Mar. 15, 1990].

Bi-functional plasmids for virus and DNA vaccines. A preferred aspect ofthe present invention concerns engineering of bi-functional plasmidsthat can serve as a DNA vaccine and a recombinant virus vector. Directinjection of the purified plasmid DNA, i.e., as a DNA vaccine, wouldelicit an immune response to the antigen expressed by the plasmid intest subjects. The plasmid would also be useful in live, recombinantviruses as immunization vehicles.

The bi-functional plasmid of the invention provides a heterologous gene,or an insertion site for a heterologous gene, under control of twodifferent expression control sequences: an animal expression controlsequence, and a viral expression control sequence. The term "undercontrol" is used in its ordinary sense, i.e., operably or operativelyassociated with, in the sense that the expression control sequence, suchas a promoter, provides for expression for expression of a heterologousgene. In a preferred embodiment, the animal expression control sequenceis a mammalian promoter (avian promoters are also contemplated by thepresent invention); in a specific embodiment, the promoter iscytomegalovirus immediate early (CMV) promoter (see FIG. 7). In afurther specific embodiment, the virus promoter is a vaccinia virusearly promoter, or a vaccinia virus late promoter, or preferably both(FIG. 7). Subjects could be vaccinated with a multi-tiered regimen, withthe bi-functional plasmid administered as DNA and, at a different time,but in any order, as a recombinant virus vaccine. The inventioncontemplates single or multiple administrations of the bi-functionalplasmid as a DNA vaccine or as a recombinant virus vaccine, or both.This vaccination regimen may be complemented with administration ofrecombinant protein vaccines (infra), or may be used with additionalvaccine vehicles.

As one of ordinary skill in the art can readily appreciate, thebi-functional plasmids of the invention can be used as polyenv vaccinevectors. Thus, by inserting at least 4 to about 10,000, preferably 4 to1000, and more preferably 10 to 100, different HIV env genes intobi-functional plasmids, thus preparing a corresponding set ofbi-functional plasmids useful as a polyenv vaccine.

Recombinant protein vaccines. Active immunity elicited by vaccinationwith an HIV env protein or proteins according to the present inventioncan prime or boost a cellular or humoral immune response. The HIV envprotein or proteins, or antigenic fragments thereof, can be prepared inan admixture with an adjuvant to prepare a vaccine.

The term "adjuvant" refers to a compound or mixture that enhances theimmune response to an antigen. An adjuvant can serve as a tissue depotthat slowly releases the antigen and also as a lymphoid system activatorthat non-specifically enhances the immune response (Hood et al.,Immunology, Second Ed., 1984, Benjamin/Cummings: Menlo Park, Calif., p.384). Often, a primary challenge with an antigen alone, in the absenceof an adjuvant, will fail to elicit a humoral or cellular immuneresponse. Adjuvants include, but are not limited to, complete Freund'sadjuvant, incomplete Freund's adjuvant, saponin, mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum. Selection of an adjuvant depends on the subject to bevaccinated. Preferably, a pharmaceutically acceptable adjuvant is used.For example, a vaccine for a human should avoid oil or hydrocarbonemulsion adjuvants, including complete and incomplete Freund's adjuvant.One example of an adjuvant suitable for use with humans is alum (aluminagel). In a specific embodiment, infra, recombinant HIV env protein isadministered intramuscularly in alum. Alternatively, the recombinant HIVenv protein vaccine can be administered subcutaneously, intradermally,intraperitoneally, or via other acceptable vaccine administrationroutes.

Vaccine administration. According to the invention, immunization againstHIV can be accomplished with a recombinant viral vaccine of theinvention alone, or in combination with a DNA vaccine or a recombinantprotein vaccine, or both. In a specific embodiment, recombinant HIV envprotein in alum is provided i.m. to boost the immune response.

Each dose of virus vaccine may contain the same 4 to 10,000, preferably4 to 1000, and more preferably 10 to 100, different recombinant viruses,each expressing a different HIV env gene. Alteratively, the viruses insubsequent vaccines may express different HIV env genes. In yet anotherembodiment, the subsequent polyenv viral vaccines may have some virusesin common, and others that are different, from the earlier vaccine. Forexample, the priming vaccine may contain vaccinia viruses expressing HIVenv proteins arbitrarily designated 1-10. A second (booster) vaccine maycontain vaccinia (or preferably a different virus, such as canarypox oradenovirus) viruses expressing HIV env proteins 6-15 or 11-20, etc.

A DNA vaccine or recombinant protein vaccine may have single HIV envprotein antigen, or multiple antigens. Preferably, a DNA or recombinantprotein vaccine for use in the invention comprises more than one HIV envprotein antigen. As with subsequent viral vaccines, the HIV env proteinor protein of a DNA vaccine or recombinant protein vaccine maycorrespond to an HIV env protein expressed in the polyenv viral vaccine,or it may be different from any of the polyenv env proteins.

In general, a preferred embodiment of the invention contemplatesproviding the greatest variety possible in each vaccination protocol, toexpose the recipient to the largest number of HIV env proteins and thusprovide the greatest opportunity for neutralizing cross-reactivity witha naive HIV isolate.

Envelope Protein Variants

As noted above, an EPV for use in the vaccines of the invention can beobtained from geographically local isolates, or clades, or fromgeographically diverse isolates, i.e., different clades. As can bereadily appreciated by one of skill in the art, obtaining envnucleotides (i.e., genes) from natural isolates has numerous advantages:the isolates are readily available, the EVPs correspond to naturallyoccurring proteins to which immunity is desirable, and mutations of HIVcan be captured quickly from new isolates.

An EPV also includes polypeptides having immunogenic activity elicitedby an amino acid sequence of an EPV amino acid sequence as at least oneepitope or antigenic determinant. This amino acid sequence substantiallycorresponds to at least one 10-900 amino acid fragment and/or consensussequence of a known HIV EPV. Such an EPV can have overall homology oridentity of at least 50% to a known envelope protein amino acidsequence, such as 50-99% homology, or any range or value therein, whileeliciting an immunogenic response against at least one strain of an HIV.

Percent homology can be determined, for example, by comparing sequenceinformation using the GAP computer program, version 6.0. available fromthe University of Wisconsin Genetics Computer Group (UWGCG). The GAPprogram utilizes the alignment method of Needleman and Wunsch [J. Mol.Biol. 48:443 (1970)], as revised by Smith and Waterman [Adv. Appl. Math.2:482 (1981)]. Briefly, the GAP program defines similarity as the numberof aligned symbols (i.e., nucleotides or amino acids) which are similar,divided by the total number of symbols in the shorter of the twosequences. The preferred default parameters for the GAP program include:(1) a unitary comparison matrix (containing a value of 1 for identitiesand 0 for non-identities) and the weighted comparison matrix of Gribskovand Burgess, Nucl. Acids Res. 14:6745 (1986), as described by Schwartzand Dayhoff, eds., Atlas of Protein Sequence and Structure, NationalBiomedical Research Foundation, Washington, D.C. (1979), pp. 353-358;(2) a penalty of 3.0 for each gap and an additional 0.10 penalty foreach symbol in each gap; and (3) no penalty for end gaps.

In a preferred embodiment, an EPV of the present invention is a variantform of at least one HIV envelope protein. Preferably, the EPV includesgp120 and the oligomerization domain of gp41, as gp140 [Hallenberger, etal., Virology 193:510-514 (1993)], entirely incorporated herein byreference).

Known HIV envelope proteins contain about 750 to 900 amino acids.Examples of such sequences are readily available from commercial andinstitutional HIV sequence databases, such as GENBANK, or as publishedcompilations, such as Myers et al., eds., Human Retroviruses and AIDS, ACompilation and Analysis of Nucleic Acid and Amino Acid Sequences, Vol.I and II, Theoretical Biology and Biophysics, Los Alamos, N.M. (1993).Substitutions or insertions of an EPV to obtain an additional EPV,encoded by a nucleic acid for use in a recombinant virus or polyenvvaccine of the present invention, can include substitutions orinsertions of at least one amino acid residue (e.g., 1-25 amino acids).Alternatively, at least one amino acid (e.g., 1-25 amino acids) can bedeleted from an EPV sequence. Preferably, such substitutions, insertionsor deletions are identified based on sequence determination of envelopeproteins obtained by nucleotide sequencing of at least one EPV encodingnucleic acid from an individual infected with HIV.

Non-limiting examples of such substitutions, insertions or deletionspreferably are made by the amplification of env DNA or RNA sequencesfrom HIV-1 infected patients, which can be determined by routineexperimentation to provide modified structural and functional propertiesof an envelope protein or an EPV. The EPVs so obtained preferably havedifferent antigenic properties from the original EPV. Such antigenicdifferences can be determined by suitable assays, e.g., by testing witha panel of monoclonal antibodies specific for HIV envelope proteins inan ELISA assay.

Any substitution, insertion or deletion can be used as long as theresulting EPV protein elicits antibodies which bind to HIV envelopeproteins, but which EPV has a different pattern than antibodies elicitedby a second EPV. Each of the above substitutions, insertions ordeletions can also include modified or unusual amino acid, e.g., asprovided in 37 C.F.R. § 1.822(p)(2), which is incorporated herein byreference.

The following Table 1 presents non-limiting examples of alternativevariants of envelope proteins of HIVs, that can be encoded by arecombinant virus according to present invention.

                                      TABLE I                                     __________________________________________________________________________    HIV Envelope Protein Variants                                                 __________________________________________________________________________                        10                  20                  30                   1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9                  __________________________________________________________________________    1    K E Q K T V A M R V K E S Q M K K       Q                                                                             H                                                                             L                                                                             W R W G W        R                                                                    W        G                                                                    T                           E   K       M     K A M G T R R N C       P                                                                             N                                                                             W                                                                             L K I   T        K                                                                    G        Y                                                                    I                                             T   T M I K K S Y       N                                                                             C                                                                             R                                                                             K G K            M                                                                    L        L                                                                    M                                             I   R   M G G E W       R                                                                             R                                                                             K I              T                                                                    T        Y                                            K E T   D       W                                                                             Q                                                                             S S              I               __________________________________________________________________________                        40                  50                  60                   1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9                  __________________________________________________________________________    31 M L L G L M I C S   A T E K L W V T       V                                                                             Y                                                                             Y                                                                             G V P V W        K                                                                    E        A                                                                    T                           L I F W I I T 6 L   V V S Q   Y A         S i   I          E                                                                    D        E                  A M A I M T P L     G A Q D               A                H                                                                    V                           I A M L T P C       I E D N                                N                    T I A             N K V                                  A               __________________________________________________________________________                        70                  80                  90                   1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9                  __________________________________________________________________________    61 T T L F C A S D A K A Y D T E V H N       V                                                                             W                                                                             A                                                                             T H A C V        P                                                                    T        D                                                                    P                           P V           E R R T H S R   A   K       I                                                                             C                                                                             S Y              N                                    N S T K A   R           K Q              G                                      L   A K   Q                                                                       E P   K                                            __________________________________________________________________________                        100                 110                 120                  1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9                  __________________________________________________________________________    91 N P Q E V V L V N V T E N F N M W K       N                                                                             D                                                                             M                                                                             V E Q M H        E                                                                    D        I                                                                    I                           D   H   I L M G S     G E   D I   R       N                                                                             I D     Q        T                                                                    V                           S   R   L Y   E       D K         T       S N                                 T   Y   M D   P         D                 Y                                           F S             H                                                  __________________________________________________________________________                        130                 140                 150                  1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9                  __________________________________________________________________________    121                                                                              S L W D Q S L K P C V K L T P L C V       S                                                                             L                                                                             K                                                                             C T D L K        N                                                                    D        T                                                                    N                           N     E E           E V M   L C V         T                                                                             M                                                                             N K H V T        T                                                                    A        S                                                                    E                                                 Q                   N                                                                             D I N Y G        G                                                                    M        T                                                            Q                                                                             S H Q W R        I                                                            I G K F L        S               __________________________________________________________________________                        160                 170                 180                  1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9                  __________________________________________________________________________    151                                                                              T S N N V T S S S W G R N I M E E G       E                                                                             I                                                                             K                                                                             N C S F N        I                                                                    S        T                                                                    S                           N K S S K   T T K N W K R E I D R E       K                                                                             M                                                                             T                                                                             K   P Y K        V                                                                    T        K                                                                    G                           I E         N V T I S K E K T G Q A       G                                                                             V                                                                             R   T   Y        Q                                                                    P        N                                G   S Q W V   I   G S       R                                                                             R                                                                             Q   E   Q        M                                                                    I                                             L   G T   V   N K       L                T                                                                    E                        __________________________________________________________________________                        190                 200                 210                  1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9                  __________________________________________________________________________    181                                                                              I R G K V Q K E Y A F F Y K L D I I       P                                                                             I                                                                             D                                                                             K G N D S        N                                                                    D                           L G D R I K Q D N S L L R N H   V V       Q                                                                             V                                                                             K                                                                             D S D I N        P                                                                    K        D                                                                    A                           V K & Q M H R V R T Y   H R T   L A       K                                                                             L                                                                             G                                                                             N                T                                                                    S                           R S   E K E T A S   T   N T P     M       E                                                                             E                                                                             G                S                  K T     Q     G H   H     V S     S       N                                                                             N                                __________________________________________________________________________                        220                 230                 240                  1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9                  __________________________________________________________________________    211                                                                              T       T S Y K   F T L T S C N T S       V                                                                             I                                                                             T                                                                             Q A C P K        V                                                                    S        F                                                                    E                           S T T N A N   T W K R I I H   S R T       T                                                                             V                                                                             K     S          I                                                                    T        Q                    S N I       R N Y I     N   D S         A                                                                             L                T                                                                    D                           S             G     K     T               I                                                       M                                                      __________________________________________________________________________                        250                 260                 270                  1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9                  __________________________________________________________________________    241                                                                              P I P I H Y C A P A G F A I L K C N       N                                                                             K                                                                             T                                                                             F N G T G        P                                                                    C        T                                                                    N                               F M   F   T G T   Y V M F     K       D                                                                             A                                                                             K S   K E        Q                                                                    K                                     H               L       R       S                                                                             P                                                                             E E   S          S                                                                    H                                                                     E                                                                             C                T                                                                    S                                                                     T                                                                             Q                I                                                                    R                        __________________________________________________________________________                        280                 290                 300                  1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9                  __________________________________________________________________________    271                                                                              V S T V Q C T H G I R P V V S T Q L       L                                                                             L                                                                             N                                                                             G S L A E        E                                                                    E        V                                                                    V                           I T S R T           K   I   T   H         I T   S K        G                                                                    G        I                                                                    K                               V   H           S   T                 S       R        K                                                                    R        D                                                                    R                                                                                      R                                                                    K        G                                                                    I                                                                                      D                                                                    M                        __________________________________________________________________________                        310                 320                 330                  1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9                  __________________________________________________________________________    301                                                                              I R S A N F T D N A K T I I V Q L N       Q                                                                             S                                                                             V                                                                             E I N C T        R                                                                    P        N                                                                    N                           L M G D D I S N S V R I W L A H   K       E                                                                             P                                                                             I                                                                             A V V Y I        E                                                                    S        I                  V   A E   L M E G T D N   V   T   T       A                                                                             T                                                                             L                                                                             Q   T   A        A                                                                    K                           M   V S   P A     G   V                   D                                                                             A                                                                             V   M   E        E                                                                    Y                                 K   K L     H                       T                                                                             T       H        H                                                                    Q                        __________________________________________________________________________                        340                 350                 360                  1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9                  __________________________________________________________________________    331                                                                              N T R K S I R   I Q R G F G R A F V       T                                                                             T                                                                             G                                                                             K I L G N        M                                                                    R        Q                                                                    A                           K V N R R   Y H R H I A P K Q V I H       A                                                                             T                                                                             R                                                                             R K I S D        I                                                                    G        K                  Y K S G N   Y K M P S   S R K T W Y       V                                                                             R                                                                             K                                                                             Q S R A N        L                                                                    L                           T R P Q T   H   L Y     L M M S V F       R                                                                             L                                                                             D                                                                             D G V F T        S                                                                    R                           S I V   G   P               S W Y I       N                                                                             M                                                                             E                                                                             A V A N I        T                                                                    V                        __________________________________________________________________________                        370                 380                 390                  1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9                  __________________________________________________________________________    361                                                                              H C N I S R A K W N N T L K Q I D S       K                                                                             L                                                                             R                                                                             E Q F G N        N                                                                    K        T                                                                    I                           Y   K L A G E Q   K A V I E G V V K       S                                                                             Y                                                                             K                                                                             K K Y K D        Q                                                                    S        V                      T V N K T D   S K A V Q K L A T       Q                                                                             Q                                                                             A H L D          H                                                                    T                                 Y   E R N   E R I   S R T   E       H                                                                             G                                                                             V R S            M                            A S A   F D     N L     R       I                                                                             I                                                                             D                                __________________________________________________________________________                        400                 410                 420                  1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9                  __________________________________________________________________________    391                                                                              I F K Q S S G G D P E I V T H S F N       C                                                                             G                                                                             G                                                                             E F F Y C        N                                                                    S        T                                                                    Q                           V S N H H A C C   L   V T M Y N L I       V                                                                             R                                                                             D I              D                                                                    T        S                                                                    G                           N L T S P         I     S L L T   T       V                A                                                                    A        N                  A   A K G         V       H   M           W                R                                                                    P                           K   S N T         Q           H           E                K               __________________________________________________________________________                        430                 440                 450                  1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9                  __________________________________________________________________________    421                                                                              L F N S T W F N S T W S T K G S N N       T                                                                             E                                                                             G                                                                             S D T I T        L                                                                    P        C                                                                    R                           M D S N I Y R L N K A G I E W N S G       M                                                                             K                                                                             E                                                                             N N N L I        H                                                                    Q        K                  I   D T   C N   V G D D P I K D G D       G                                                                             G                                                                             R                                                                             E G P V V        I                                                                    L                               T G   F 8   D S K K N T C G T S       N                                                                             Q                                                                             A                                                                             R E L   K        D                        A     G       M   G   M L D I       Q                                                                             S                                                                             K R S                            __________________________________________________________________________                        460                 470                 480                  1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9                  __________________________________________________________________________    451                                                                              I K Q I I N M W Q E V G K A M Y A P       P                                                                             I                                                                             S                                                                             G Q I R C        S                                                                    S        N                                                                    I                               E F V R I   A G T R Q S T   D L       F                                                                             G                                                                             R V L S F        I                            K R     R A   R   L             T                                                                             Q E   K          E                              S     K         I             K T   T          V                              L     V                       E L              T               __________________________________________________________________________                        490                 500                 510                  1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9                  __________________________________________________________________________    481                                                                              T G L L L T R D G G A N E N N E S E       I                                                                             F                                                                             R                                                                             P G G G D        M                                                                    R        D                                                                    N                               T I   V S   S V T D Q T S D T V       V                                                                             I                                                                             S                                                                             L T     N        I                                                                    K        N                                                                    I                               I     E         E S K S A G E N       T                                                                             L A     E                                               D G T A K R N L         V                                                     G E D K   T I           I                              __________________________________________________________________________                        520                 530                 540                  1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9                  __________________________________________________________________________    511                                                                              W R S E L Y K Y K V V K I E P L G V       A                                                                             P                                                                             T                                                                             K A K R R        V                                                                    V        Q                                                                    R                           R I N K   F N     D I R V K L I S I       S                                                                             R S R   P        I                                                                    M        E                      T                 T     T F             P S   H        I                                                                    A                                                 Q                     M A            W                                        E                     I              H               __________________________________________________________________________                        550                 560                 570                  1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9                  __________________________________________________________________________    541                                                                              E K R A V G E I G A L F L G F L G A       A                                                                             G                                                                             S                                                                             T M G A A        S                                                                    M        T                                                                    L                           K E     I F I V   V M S I   V   S         S   A   V        A                                                                    L        A                                                                    V                           Q       A V T L*  M V L P                         R        P                                                                    I                                       A M     F I                           G        V                              L         I                                    T                                                                    T                        __________________________________________________________________________                        580                 590                 600                  1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9                  __________________________________________________________________________    571                                                                              T V Q A R Q L L S G I V Q Q Q N N L       L                                                                             R                                                                             A                                                                             I E A Q Q        H                                                                    L        L                                                                    Q                           A G R T H H V M K D     H     S           M K G            Q                                                                    M        K                      P P   L                   D           R D              E                        L   K                                 Q              R                            S                                                                __________________________________________________________________________                        610                 620                 630                  1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9                  __________________________________________________________________________    601                                                                              L T V W G I K Q L Q A R I L A V E R       Y                                                                             L                                                                             K                                                                             D Q Q L L        G                                                                    I        W                                                                    G                             S I     V R     R L   V Q   L   T       F                                                                             I                                                                             R                                                                             E   R R M        E                                                                    F        L                                                                    W                                               T   L     I   S       L                                                                             Q                                                                             N   K I          R                                                                    M                                                                     G                S                                                                    N                                                                                      N                                                                    L                        __________________________________________________________________________                        640                 650                 660                  1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9                  __________________________________________________________________________    631                                                                              C S G K L I C T T A V P W N A S W S         N                                                                             K                                                                             S                                                                             L E Q I W      N                                                                      N      M T                R K   R T V   P   T   K     S T   G         R                                                                             R                                                                             T                                                                             M D D F        D                                                                      K      T M                        H     Y   N         F A   S         Y                                                                             N                                                                             Q N M          G                                                                      H      L                          I         F         N               G                                                                             V S S          Q                                                                      T      N                          A         S                         R K K          W               __________________________________________________________________________                        670                 680                 690                  1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9                  __________________________________________________________________________    661                                                                              W M E W D R E I N N Y T S L I H S L       I                                                                             E                                                                             E                                                                             S Q N Q Q        E                                                                    K        N                                                                    E                             L Q   E K L V D S V S N T   Y T I       L                                                                             T                                                                             D                                                                             A A I            G                                                                    I        Q                    I K     Q H   E K I   G I   F N E       Q                                                                             Q   T            D                                                                    Q        V                    Q         Q   S       D V               N   D            R                                          K E                   V                            __________________________________________________________________________                        700                 710                 720                  1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9                  __________________________________________________________________________    691                                                                              Q E L L E L D K W A S L W N W F N I       T                                                                             N                                                                             W                                                                             L W Y I K        L                                                                    F        I                                                                    M                           L D     G   N E   T N     S   S S         S                                                                             Q   S   R        I                                                                    A        V                                                                    I                           R A     A     S   K G         Y G         K                                   K K     K     Q               L D                                                     Q                                                                  __________________________________________________________________________                        730                 740                 750                  1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9                  __________________________________________________________________________    721                                                                              I V G G L V G L R I V F A V L S V V       N                                                                             R                                                                             V                                                                             R Q G Y S        P                                                                    L        S                                                                    F                           V I A A I I   V K V I M S I F C I I       K                                                                             S                                                                             F                                                                             S A     Q        L                  A                       T     N L         R                                                                             N                                                                             I                                                                             N                                                           I                                                  __________________________________________________________________________                        760                 770                 780                  1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9                  __________________________________________________________________________    751                                                                              Q T H L P I P R G P D R P E G I E E       E                                                                             G                                                                             G                                                                             E R D R D        R                                                                    S        I                                                                    R                             I R T H V Q E E L G Q L D R T D G       G                                                                             D Q G K G        T                                                                    W        V                                                                    G                               L A N T T G   A E T Q G E               G     P        G                                                                    G        Q                      P P I A R Q                             E     S        K                                                                    N        P                        F   G S                                                                                                                            S                                                                    A                        __________________________________________________________________________                        790                 800                 810                  1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9                  __________________________________________________________________________    781                                                                              L V N G S L A L I W D D L R S L C L       F                                                                             S                                                                             Y                                                                             H R L R D        L                                                                    L        L                                                                    I                           A L D   F S T Q F Y E   C W T C F S       S                                                                             C                                                                             R L   T N        F                                                                    A        S                                                                    T                           S P H   L   P   L   V     G N I I I       W                                                                             L                                                                             Q S   S S        C                                                                    I        C                                                                    V                               T   C   Q       G       A   G         T                Q                      S               T                                      H               __________________________________________________________________________                        820                 830                 840                  1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9                  __________________________________________________________________________    811                                                                              V T R I V E L L G R R G W E A L K Y       W                                                                             W                                                                             N                                                                             L L Q Y W        S                                                                    Q        E                                                                    L                           A A K T I D I   K H G L L D G I R L       L                                                                             G                                                                             S                                                                             V V L            I                                                                    K                           I V   A L S T   R L L I   N I C   I       C                                                                             A                                                                             A M I            G                                                                    R                             K   L   K Y               V             G                                                                             C T              T                    M   V                     R                              L               __________________________________________________________________________                        850                 860                 870                  1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9                  __________________________________________________________________________    841                                                                              K N S A V S L L N A T A I A V A E G       T                                                                             D                                                                             R                                                                             V I E V V        Q                                                                    G        A                                                                    Y                           R I   V I N W F D T I   V V   T G E       G                                                                             I   L I A        R                                                                    R        I                                                                    C                           Q S   F S   F V A   V     S     N R       K                                                                             A   A G          A                                                                    T        L                          T           L             W             A          T                                                                    V        G                                                                             F                                                                    V                        __________________________________________________________________________                        880                 889                                   __________________________________________________________________________       1 2 3 4 5 6 7 8 9   1 2 3 4 5 6 7 8 9                                      __________________________________________________________________________    871                                                                              R A I R H I P R R I R Q G L E R I L      L                                    Q G F L N V H T   V       F K G L        Q                                    T I V I                   A     A        V                                                                    R                                                                             S                                          __________________________________________________________________________

Accordingly, based on the above examples of specific substitutions,alternative substitutions can be made by routine experimentation, toprovide alternative EPVs of the present invention, e.g., by making oneor more substitutions, insertions or deletions in envelope proteins orEPV's which give rise to differential immune responses.

Amino acid sequence variations in an EPV of the present invention can beprepared e.g., by mutations in the DNA. Such EPVs include, for example,deletions, insertions or substitutions of nucleotides coding fordifferent amino acid residues within the amino acid sequence. Obviously,mutations that will be made in nucleic acid encoding an EPV must notplace the sequence out of reading frame and preferably will not createcomplementary domains that could produce secondary mRNA structures [see,e.g., Ausubel (1995 rev.), infra; Sambrook (1989), infra].

EPV-encoding nucleic acid of the present invention can also be preparedby amplification or site-directed mutagenesis of nucleotides in DNA orRNA encoding an envelope protein or an EPV, and thereafter synthesizingor reverse transcribing the encoding DNA to produce DNA or RNA encodingan EPV [see, e.g., Ausubel (1995 rev.), infra; Sambrook (1989), infra],based on the teaching and guidance presented herein.

Recombinant viruses expressing EPV's of the present invention,recombinant EPVs, or nucleic acid vectors encoding therefor, include afinite set of EPV-encoding sequences as substitution nucleotides thatcan be routinely obtained by one of ordinary skill in the art, withoutundue experimentation, based on the teachings and guidance presentedherein. For a detailed description of protein chemistry and structure,see Schulz, G. E. et al., Principles of Protein Structure,Springer-Verlag, New York, N.Y. (1978), and Creighton, T. E., Proteins:Structure and Molecular Properties, W. H. Freeman & Co., San Francisco,Calif. (1983), which are hereby incorporated by reference. For apresentation of nucleotide sequence substitutions, such as codonpreferences, see Ausubel et al., eds, Current Protocols in MolecularBiology, Greene Publishing Assoc., New York, N.Y. (1987, 1988, 1989,1990, 1991, 1992, 1993, 1994, 1995) (hereinafter, "Ausubel (1995 rev.)")at §§ A.1.1-A.1.24, and Sambrook, J. et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1989) at Appendices C and D.

Thus, one of ordinary skill in the art, given the teachings and guidancepresented herein, will know how to substitute other amino acid residuesin other positions of an env DNA or RNA to obtain alternative EPVs,including substitutional, deletional or insertional variants.

Screening Assays for HIV Activity

For screening anti-HIV activity of sera or cells from an individualimmunized with a vaccine of the invention, any known and/or suitablescreening assay can be used, as is known in the art. For example, knownHIV assays include viral infectivity assays [see, e.g., Chesebro et al.,J. Virol. 62:3779-3788 (1988); Aldovini et al., eds., Techniques in HIVResearch pp. 71-76 (1990)]; neutralization assays [see, e.g., Golding etal., AIDS Res. Hum. Retrovir. 10:633-643 (1994); Hanson., AIDS Res. Hum.Retrovir. 10:645-648 (1994); Laal et al., Res. Hum. Retrovir. 9:781-785(1993); Hanson, J. Acquir. Immune Defic. Syndr. 7:211-219 (1994)];peripheral mononuclear (PMN) cell assays [see, e.g., Arduino et al.,Antimicrob. Agents Chemother. 37:1095-1101 (1990)]; and cytotoxicT-lymphocyte (CTL) assays [see, e.g., Hammond et al., J. Exp. Med.176:1531-1542 (1992); McElrath et al., J. Virol. 68:5074-5083 (1994);Walker et al., Cell. Immunol. 119:470-475 (1989); Weinhold et al., AIDSRes. Hum. Retrovir. 8:1373 (1992)]. Other suitable activities, alone orin any combination, include, but are not limited to, quantitative and/orqualitative measurement of transcription, replication, translation,virion incorporation, virulence, viral yield, and/or morphogenesis. Theabove references are entirely incorporated herein by reference.

Specific Embodiment: Recombinant Vaccinia Virus Encoding EPV's, PolyenvVaccines and Methods of Making and Using Thereof

Overview. Recombinant vaccinia viruses (VV) expressing HIV envelopeproteins (e.g., gp 41, gp 120 and/or gp 160, or a portion thereof)provide materials useful for the production and testing of mixedvaccines that induce at least one of a humoral or cellular immuneresponse against the virus, as well as for analyses of B-cell and CTLdeterminants.

A polyenv vaccine of the present invention consists of a mixture of ndistinct recombinant vaccinia viruses, where n is a whole number fromabout 4 to about 10,000 (or any range or value therein), wherein eachvaccinia vector construct expresses a variant of a HIV-1 envelopeprotein (EPV) (e.g., gp 41, gp 120 or gp 160). The recombinant vacciniavirus functionally encodes an EPV and is prepared by recombination ofwildtype VV with a plasmid. Multiple, distinct plasmids encoding EPV canbe prepared by substituting one EPV encoding sequence with another,e.g., using a restriction fragment or mutagenesis.

Preparation of Recombinant Vaccinia Viruses. Methods for the preparationof individual plasmids (each expressing a unique HIV protein sequence)can utilize DNA or RNA amplification for the substitution of isolatedenvelope protein variant sequences into a vector (e.g., pVenv4 or pVenv1[Hallenberger et al., Virology 193:510-514 (1993)], which vector encodesa known HIV envelope protein sequence (e.g., available from the NIAIDAIDS Research & Reference Reagent Program, Rockville, Md.).

Methods of amplification of RNA or DNA are well known in the art and canbe used according to the present invention without undueexperimentation, based on the teaching and guidance presented herein.Known methods of DNA or RNA amplification include, but are not limitedto polymerase chain reaction (PCR) and related amplification processes(see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188,to Mullis et al.; U.S. Pat. Nos. 4,795,699 and 4,921,794 to Tabor et al;U.S. Pat. No. 5,142,033 to Innis; U.S. Pat. No. 5,122,464 to Wilson etal.; U.S. Pat. No. 5,091,310 to Innis; U.S. Pat. No. 5,066,584 toGyllensten et al; U.S. Pat. No. 4,889,818 to Gelfand et al; U.S. Pat.No. 4,994,370 to Silver et al; U.S. Pat. No. 4,766,067 to Biswas; U.S.Pat. No. 4,656,134 to Ringold) and RNA mediated amplification which usesanti-sense RNA to the target sequence as a template for double strandedDNA synthesis (U.S. Pat. No. 5,130,238 to Malek et al, with the tradename NASBA), the entire contents of which patents are herein entirelyincorporated by reference.

For example, recombinant vaccinia virus constructs prepared by thisroute can be used for immunizations and elicitation of HIV-specific Tand/or B-cell responses. Primers utilize conserved HIV sequences andthus successfully amplify env genes from many diverse HIV-1 patientsamples. The basic techniques described here can similarly be used withPCR or other types of amplification primers, in order to substitutesmaller or larger pieces of the env sequence from field isolates forthat found in vectors encoding an HIV envelope protein. See, e.g.,Ausubel; infra, Sambrook, infra.

EPV Encoding Nucleic Acids. The technique begins with the isolation ofDNA from HIV infected cells and the amplification of env sequences byPCR. PCR or other amplification products provide the simplest means forthe isolation of HIV sequences, but any other suitable and known methodscan be used such as cloning and isolation of EPV encoding nucleic acidor proteins (see Ausubel, infra; Sambrook, infra). Enzyme restrictionsites are preferably incorporated into PCR or other amplification primersequences to facilitate gene cloning.

Isolated DNA for PCR can be prepared from multiple virus sources,inclusive of fresh or frozen whole blood from HIV+ patients and cellsthat have been infected in vitro with virus isolates.

In order to produce new HIV env constructs, the polymerase chainreaction (PCR) is preferably used to amplify 100-2700 base pairs (bp) ofan env gene from each different HIV patient sample. The PCR primers canrepresent well-conserved HIV sequences which are suitable for amplifyingenv genes from known samples of env genes, isolated HIVs or diverse HIVpatient samples. The amplified DNA preferably comprises a portionencoding 10-900 (such as 100-400, 400-600 or 600-900, or any range orvalue therein) amino acids of a gp120 and gp41 (both make up gp160). Oneor more of the envelope variable regions (V1-V5) and constant regions(C1-C5) are preferably included in the PCR products, more preferablymost of the V1, C1, V2, C2, V3, C3, V4, C4, and V5 regions. In addition,amplified sequences can encode 1-200 amino acids beyond the cleavagesite for gp120/gp41. Preferably, most or all of the entire env gene isamplified. Optionally, the gp160 encoding sequence amplified is missingpart or all of sequences encoding the transmembrane domain and/or thecytoplasmic tail domain [see, e.g., Hallenberger et al. (1993)].

The PCR primers can be designed so that restriction enzyme sites flankthe envelope gene sequence in vaccinia plasmid, such that they areincorporated into the amplified DNA products. By using well-knownsubstitution cloning techniques, vaccinia plasmid derivatives thatexpress envelope protein variant sequences from 1-10,000 patients can begenerated by substituting a portion of the patient's EPV encodingsequence for a corresponding portion of the env sequence in the vacciniaplasmid, such as by using restriction fragments for the substitution.For example, the pVenv4 plasmid and PCR products are treated with KpnIand BsmI to obtain a sequence encoding a truncated gp160 of amino acids1-639, which lacks both the transmembrane domain and the cytoplasmictail domain of gp41[ see, e.g., Hallenberger et al. (1993)]

Following ligation of the PCR product and the pVenv products, bacterialhost cells are transformed with the ligation mixture via any of a numberof methods well-known in the art, including, e.g., electroporation, andrecombinant colonies are picked and examined by sequencing.

Recombinant Vaccinia Virus Constructs Encoding HIV Envelope Proteins.The EPV encoding vaccinia is then recombined with wild type virus in ahost cell and the EPV expressing virus plaques are selected and virusstocks made. The virus stocks as VVenv's each containing a different EPVencoding sequence are then mixed using at least 4-40, and up to about10,000 different recombinant viruses, to form a polyenv vaccine of thepresent invention.

The recombinant vaccinia plasmids containing the EPV sequences are thenoptionally sequenced or screened with HIV envelope protein-specificantibodies to identify different EPVs. Sequencing by the Sanger Methoddideoxy-chain termination is preferred. The procedure is preferablyadapted from previously described methods [Sambrook et al. (1989),infra; United States Biochemical, Sequenase Version 2.0--DNA SequencingKit, Ninth Edition, Amersham Life Science, Inc., (1994)] and should readapproximately 50-300 bp from the primer position.

Methods for the production of VV expression vectors are well-known inthe art [see, e.g., Mackett, M. et al., Proc. Natl. Acad. Sci. (USA)79:7415-7419 (1982); Panicali, D., and Paoletti, E., Proc. Natl. Acad.Sci. (USA) 79:4927-4931 (1982); U.S. Pat. No. 4,169,763; Mazzara, G. P.et al., Methods in Enz. 217:557-581 (1993), Ausubel et al., infra, at §§16.15-16.19, each of which are entirely incorporated herein byreference]. The previously described pSC11 vector [Chakrabarti, S. etal., Mol. Cell. Biol. 5:3403-3409 (1985)] can preferably be used tocreate an env-encoded plasmid, such as pVenv4.

As a viral vector, vaccinia virus has a number of usefulcharacteristics, including capacity that permits cloning large fragmentsof foreign DNA (greater than 20 Kb), retention of infectivity afterinsertion of foreign DNA, a wide host range, a relatively high level ofprotein synthesis, and suitable transport, secretion, processing andpost-translational modifications as dictated by the primary structure ofthe expressed protein and the host cell type use. For example,N-O-glycosylation, phosphorylation, myristylation, and cleavage, as wellas assembly of expressed proteins, occur in a faithful manner.

Several variations of the vaccinia vector have been developed and aresuitable for use in the present invention (e.g., see Ausubel et al.,infra, §§ 16.15-16.19). Most commonly, after obtaining the virus stock(Ausubel, infra at § 16.16), a nucleic acid sequence encoding an EPV isplaced under control of a vaccinia virus promoter and integrated intothe genome of vaccinia so as to retain infectivity (Ausubel et al.,infra at § 16.17). Alternatively, expression can be achieved bytransfecting a plasmid containing the vaccinia promoter-controlled geneencoding an EPV into a cell that has been infected with wild-typevaccinia.

Preferably, the host cell and vaccinia vector are suitable and approvedfor use in vaccination of mammals and humans. These recombinant virusesare then characterized using various known methods (Ausubel et al.,infra at § 16.18). In still another variation, the bacteria phage T7 RNApolymerase chain can be integrated into the genome of vaccinia so thatthe EPV encoding sequences will be expressed under the control of a T7promoter, either in transfected plasma, plasmid or a recombinantvaccinia virus, will be expressed.

The use of pox virus promoters is preferred because cellular and otherviral promoters are not usually recognized by the vacciniatranscriptional apparatus. A compound early/late promoter is preferablyused in recombinant vaccinia for polyenv vaccines, as it is desirable toexpress the EPV as an antigen that is presented in recombinant vacciniavirus infected host cell in association with major histocompatibilityclass (MHC) I or II. Such MHC associated HIV envelope protein will thenform cytotoxic T cell targets, and prime vaccinated mammals for acytotoxic T cell response and/or a humoral response against theexpressed HIV EPVs. This is because the ability of vaccinia viralvectors to induce MHC presentation in host cells for this type ofantigen appears to diminish late in the infection stage. Transcriptsoriginating early will terminate after the sequence TTTTTNT and lead toinadequate MHC presentation.

Alternatively, any such termination motifs within the coding sequence ofthe gene can be altered by mutagenesis if an early pox virus promoter isused, in order to enhance MHC presentation of envelope protein antigensin host cells (Earl et al., infra, 1990). To mimic vaccinia virus mRNAs,untranslated leader and 3'-terminal sequences are usually kept short, ifthey are used in the vaccinia plasmids incorporating HIV EPV encodingsequences.

Preferably, the plasmid used for making vaccinia constructs according tothe present invention has been designed with restriction endonucleasesites for insertion of the env gene downstream of the vaccinia promoter(Ausubel et al., infra, § 16.17). More preferably, the plasmid alreadycontains an envelope protein encoding sequence, wherein the restrictionsites occur uniquely near each of the beginning and ends of the envelopeprotein coding sequence. The same restriction fragment of the EPVencoding sequence can then replace the corresponding sequence in theplasmid. In such cases, the major portion of the EPV encoding sequencecan be inserted after removing most or all of the envelope proteinencoding sequence from the plasmid.

Preferably, the resulting vaccinia construct (containing the EPVencoding sequence and the vaccinia promoter) is flanked by vaccinia DNAto permit homologous recombination when the plasmid is transfected intocells that have been previously infected with wild-type vaccinia virus.The flanking vaccinia virus DNA is chosen so that the recombination willnot interrupt an essential viral gene.

Without selection, the ratio of recombinant to parental vaccinia virusis usually about 1:1000. Although this frequency is high enough topermit the use of plaque hybridization (see Ausubel et al., infra at §§6.3 and 6.4) or immunoscreening (Ausubel et al., infra at § 6.7) to pickrecombinant viruses, a variety of methods to facilitaterecombinant-virus identification have been employed. Nonlimitingexamples of such selection or screening techniques are known in the art(see Ausubel et al., infra at § 16.17). Usually, the expression cassetteis flanked by segments of the vaccinia thymidine kinase (TK) genes sothat recombination results in inactivation of TK. Virus with a TK⁻phenotype can then be distinguished from those with a TK⁺ phenotype byinfecting a TK⁻ cell line in the presence of 5-bromo-deoxyuridine(5-BrdU), which must be phosphorylated by TK to be lethally incorporatedinto the virus genome. Alternatively or additionally, recombinantviruses can be selected by the co-expression of a bacterial antibioticresistant gene such as ampicillin (amp) or guanine phosphoribosyltransferase (gpt). As a further example, co-expression of theEscherichia coli lac Z gene allows co-screening of recombinant virusplaques with Xgal (Ausubel, infra, § 16.17).

The recombinant vaccinia viruses expressing an EPV of the presentinvention can be optionally attenuated or inactivated according to knownmethods, such as by heat, paraformaldehyde treatment, ultravioletirradiation, propriolactene treatment, hybrid or chimera formation or byother known methods [see, e.g., Zagury et al., Nature 332:728-731(1988); Ito et al., Cancer Res. 50:6915-6918 (1990); Wellis et al., J.Immunol. 99:1134-9 (1967); D'Honcht, Vaccine 10 (Suppl.):548-52 (1992);Selenka et al., Arch. Hyg. Bakteriol. 153:244-253 (1969);Grundwald-Bearch et al., J. Cancer Res. Clin. Oncol. 117:561-567 (1991);the contents of which are entirely incorporated here by reference]. Forexample, heat inactivation at 60° C. will reduce virus titerconsiderably. Such attenuation techniques are safety tested, asincomplete inactivation might result in patient death [Dorozynski andAnderson, Science 252:501-502 (1991)].

Such attenuated or inactivated recombinant vaccinia is to be used wherethe patient may have a compromised immune system as complications ordeath can occur when live vaccinia is administered.

Pharmaceutical Compositions

Pharmaceutical preparations of the present invention, suitable forinoculation or for parenteral or oral administration, include a polyenvrecombinant virus vaccine comprising of at least 4, and up to about10,000, preferably 4 to about 1000, and more preferably about 10 toabout 100 different recombinant viruses, in the form of a cell lysate,membrane-bound fraction, partially purified, or purified form.Preferably, the polyenv vaccine comprises recombinant virus containingcell lysate (or membrane-bound fractions thereof) that further compriseEPV proteins already expressed by the recombinant viruses. The inclusionof the expressed EPVs is now discovered to enhance the primary antibodyresponse.

The polyenv vaccine composition can be in the form of sterile aqueous ornon-aqueous solutions, suspensions, or emulsions, and can also containauxiliary agents or excipients which are known in the art. Each of theat least about 4-40 to 10,000 different viruses encode and express adifferent EPV, as presented herein. EPVs encoding DNA can be selected torepresent EPVs existing in a specific isolated community of AIDSpatients. For example, a vaccine could represent sequences from Memphis,Tenn. and be targeted for use in Memphis, Tenn. Vaccines designed torepresent geographically restricted areas can also be useful for use incommunities outside of the targeted community.

Alternatively, EPVs encoding DNAs can be selected to representgeographically distant communities, cities or countries, such as clades.For example, multiple clones can be represented in one polyenv vaccine.A polyenv vaccine composition can further comprise immunomodulators suchas cytokines which accentuate an immune response to a viral infection.See, e.g., Berkow et al., eds., The Merck Manual, Fifteenth Edition,Merck and Co., Rahway, N.J. (1987); Goodman et al., eds., Goodman andGilman's The Pharmacological Basis of Therapeutics, Eighth Edition,Pergamon Press, Inc., Elmsford, N.Y. (1990); Avery's Drug Treatment:Principles and Practice of Clinical Pharmacology and Therapeutics, ThirdEdition, ADIS Press, LTD., Williams and Wilkins, Baltimore, Md. (1987);and Katzung, ed. Basic and Clinical Pharmacology, Fifth Edition,Appleton and Lange, Norwalk, Conn. (1992), which references andreferences cited therein, are entirely incorporated herein by referenceas they show the state of the art.

As would be understood by one of ordinary skill in the art, when apolyenv vaccine of the present invention is provided to an individual,it can be in a composition which can further comprise at least one ofsalts, buffers, adjuvants, or other substances which are desirable forimproving the efficacy of the composition. Adjuvants are substances thatcan be used to specifically augment at least one immune response.Normally, the adjuvant and the composition are mixed prior topresentation to the immune system, or presented separately, but into thesame site of the being immunized. Adjuvants can be loosely divided intoseveral groups based upon their composition. These groups include oiladjuvants, mineral salts (for example, AlK(SO₄)₂, AlNa(SO₄)₂, AlNH₄(SO₄), silica, kaolin, and carbon), polynucleotides (for example, polyIC and poly AU nucleic acids), and certain natural substances (forexample, wax D from Mycobacterium tuberculosis, substances found inCorynebacterium parvum, or Bordetella pertussis, and members of thegenus Brucella). Among those substances particularly useful as adjuvantsare the saponins (e.g., Quil A., Superfos A/S, Denmark). Examples ofmaterials suitable for use in vaccine compositions are disclosed, e.g.,in Osol, A., ed., Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pa. (1980), pp. 1324-1341, which reference is entirelyincorporated herein by reference.

A pharmaceutical polyenv vaccine composition of the present inventioncan further or additionally comprise at least one antiviralchemotherapeutic compound. Non-limiting examples can be selected from atleast one of the group consisting of gamma globulin, amantadine,guanidine, hydroxy benzimidazole, interferon-α, interferon-β,interferon-γ, interleukin-16 (IL-16; Kurth, Nature, Dec. 8, 1995);thiosemicarbarzones, methisazone, rifampin, ribvirin, a pyrimidineanalog (e.g., AZT and/or 3TC), a purine analog, foscarnet,phosphonoacetic acid, acyclovir, dideoxynucleosides, a proteaseinhibitor (e.g., saquinavir (Hoffmann-La Roche); indinavir (Merck);ritonavir (Abbott Labs); AG 1343 (Agouron Pharmaceuticals); VX-2/78(Glaxo Wellcome)); chemokines, such as RANTES, MIP1α or MIP1β [Science270:1560-1561 (1995)] or ganciclovir. See, e.g., Richman: AIDs Res. Hum.Retroviruses 8: 1065-1071 (1992); Annu Rev Pharmacol Toxico 33: 149-164(1993); Antimicrob Agents Chemother 37: 1207-1213 (1993); AIDs Res. Hum.Retroviruses 10: 901 (1994): Katzung (1992), infra, and the referencescited therein on pages 798-800 and 680-681, respectively, whichreferences are herein entirely incorporated by reference.

Pharmaceutical Uses

The administration of a polyenv vaccine (or the antisera which itelicits) can be for either a "prophylactic" or "therapeutic" purpose,and preferably for prophylactic purposes. When providedprophylactically, the live polyenv vaccine composition is provided inadvance of any detection or symptom of HIV infection or AIDS disease.The prophylactic administration of the compound(s) serves to prevent orattenuate any subsequent HIV infection.

When provided therapeutically, the polyenv vaccine is provided upon thedetection of a symptom of actual infection. The administration of a livepolyenv vaccine after HIV infection is provided only where the patient'simmune system is determined to be capable of responding toadministration of the live polyenv vaccine without substantive risk ofunsuitable complications or death, where the administration of a livevirus is provided in the required dosage that serves to attenuate anyactual HIV infection.

Alternatively, where the patient's immune response is compromised,therapeutic administration preferentially involves the use of anattenuated or inactivated polyenv vaccine composition where therecombinant viruses are attenuated or inactivated, as presented above.See, e.g., Berkow (1987), infra, Goodman (1990), infra, Avery (1987),infra and Katzung (1992), infra, Dorozynski and Anderson, Science252:501-502 (1991) which are entirely incorporated herein by reference,including all references cited therein.

A composition is said to be "pharmacologically acceptable" if itsadministration can be tolerated by a recipient patient. Such an agent issaid to be administered in a "therapeutically or prophylacticallyeffective amount" if the amount administered is physiologicallysignificant. A vaccine or composition of the present invention isphysiologically significant if its presence results in a detectablechange in the physiology of a recipient patient, preferably by enhancinga humoral or cellular immune response to an HIV.

The "protection" provided need not be absolute, i.e., the HIV infectionor AIDS disease need not be totally prevented or eradicated, providedthat there is a statistically significant. improvement relative to acontrol population. Protection can be limited to mitigating the severityor rapidity of onset of symptoms of the disease.

Pharmaceutical Administration

A vaccine of the present invention can confer resistance to one or morestrains of an HIV. The present invention thus concerns and provides ameans for preventing or attenuating infection by at least one HIVstrain. As used herein, a vaccine is said to prevent or attenuate adisease if its administration to an individual results either in thetotal or partial attenuation (i.e. suppression) of a symptom orcondition of the disease, or in the total or partial immunity of theindividual to the disease.

At least one polyenv vaccine of the present invention can beadministered by any means that achieve the intended purpose, using apharmaceutical composition as described herein.

For example, administration of such a composition can be by variousparenteral routes such as subcutaneous, intravenous, intradermal,intramuscular, intraperitoneal, intranasal, transdermal, or buccalroutes. Subcutaneous administration is preferred. Parenteraladministration can be by bolus injection or by gradual perfusion overtime. See, e.g., Berkow (1987), infra, Goodman (1990), infra, Avery(1987), infra, and Katzung (1992), infra, which are entirelyincorporated herein by reference, including all references citedtherein.

A typical regimen for preventing, suppressing, or treating a disease orcondition which can be alleviated by a cellular immune response byactive specific cellular immunotherapy, comprises administration of aneffective amount of a vaccine composition as described is above,administered as a single treatment, or repeated as enhancing or boosterdosages, over a period up to and including one week to about 24 months.

According to the present invention, an "effective amount" of a vaccinecomposition is one which is sufficient to achieve a desired biologicaleffect, in this case at least one of cellular or humoral immune responseto HIV. It is understood that the effective dosage will be dependentupon the age, sex, health, and weight of the recipient, kind ofconcurrent treatment, if any, frequency of treatment, and the nature ofthe effect desired. The ranges of effective doses provided below are notintended to limit the invention and represent preferred dose ranges.However, the most preferred dosage will be tailored to the individualsubject, as is understood and determinable by one of skill in the art,without undue experimentation. See, e.g., Berkow (1987), infra, Goodman(1990), infra, Avery (1987), infra, Ebadi, Pharmacology, Little, Brownand Co., Boston, Mass. (1985), and Katsung (1992), infra, whichreferences and references cited therein, are entirely incorporatedherein by reference.

Generally speaking, the dosage for a human adult will be from about 10⁵-10⁹ plaque forming units (pfu)/kg or colony forming units (CFU)/kg perdose, with 10⁶ -10⁸ preferred. Whatever dosage is used, it should be asafe and effective amount as determined by known methods, as alsodescribed herein.

Subjects

The recipients of the vaccines of the present invention can be anymammal which can acquire specific immunity via a cellular or humoralimmune response to HIV, where the cellular response is mediated by anMHC class I or class II protein. Among mammals, the preferred recipientsare mammals of the Orders Primata (including humans, chimpanzees, apesand monkeys). The most preferred recipients are humans. The subjectspreferably are infected with HIV or provide a model of HIV infection[e.g., Hu et al., Nature 328:721-723 (1987)], which reference isentirely incorporated herein by reference.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples, whichare provided by way of illustration, and are not intended to be limitingof the present invention.

EXAMPLES Example 1

Preparation of Vaccinia Virus Vectors for HIV Env Protein Expression

Nomenclature. For purposes of reference, a recombinant vaccinia virusconstruct is alternatively referred to herein as a VVenv construct, withspecific vaccinia virus constructs being designated according to apatient, or to a depository (e.g., ATCC br the GenBank source of the envDNA in the construct). For example, VVenv-Doe would refer to a vacciniavirus vector construct having env sequences from patient Doe, andVVenv-U28305 would refer to a vaccinia virus vector having the envsequences found in GenBank accession No. U28305.

The polyenv vaccine consists of 4-100 distinct recombinant vacciniaviruses, each of which expresses a unique HIV-1 envelope protein. Forpurposes of reference, each individual virus is designated as VVenv, andthe final virus mixture is referred to as polyenv.

The preparation of each VVenv uses the plasmid designated pVenv4 and awildtype vaccinia virus designated NYCDH, described below. Foradditional details, see Ryan et al., "Preparation and Use of VacciniaVirus Vectors for HIV Protein Expression and Immunization," inImmunology Methods Manual, Lefkovits, ed., Academic Press (1996).

Vectors and Host Cells. The previously described pSC11 vector[Chakrabarti, S. et al., Mol. Cell. Biol. 5:3403-3409 (1985)] can beused for the recombination of multiple HIV genes into the VV genome.Elements of the pSC11 plasmid include the lacZ gene (a reporter gene bywhich transformed bacteria and VV recombinants can be easily identifiedas those having β-galactosidase activity), a portion of the geneencoding thymidine kinase (TK), and an ampicillin resistance gene (amp).Genes cloned into pSC11 are inserted into the VV genome by homologousrecombination between the TK gene of the wildtype virus and the portionsof the TK gene contained in pSC11. Insertion of plasmid DNA into theviral TK locus inactivates the viral gene so that recombinant virusescan be readily selected from the background of TK⁺ virus by growth inbromodeoxyuridine (BUdR). In order for recombinant TK⁻ virus to survivethis selection, they must be grown in cells which do not supply anactive TK enzyme, such as the TK⁻ 143 cell line, which is a TK-deficientderivative of the human cell line R970-5, an osteosarcoma cell line(Rhim, J. S. et al., Int. J. Cancer 15:23-29 (1975)] that supports thegrowth of VV [Weir et al., infra (1982)]. The production of HIV genesegment expression can be by full gene insertion into the SmaI site ofthe pSC11 vector. Full length genes can be expressed under the controlof the P7.5K promoter.

As an alternative to the cloning of complete HIV genes, one cansubstitute partial gene sequences for HIV genes that have already beencloned into pSC11. For example, a construct termed pVenv1 was preparedfrom pSC11 and expresses the BH10 HIV envelope protein (env) gene[Hallenberger et al., infra, (1993); Kilpatrick et al. J. Biol. Chem.262:116-121 (1987)]. The construct can be used as a parent vector tosubstitute and express variable envelope protein regions from field HIVisolates. Similarly, a vector termed pVenv4 was constructed from pSC11to express a BH10 env protein, truncated to exclude the transmembraneand cytoplasmic tail domain encoding gp41 sequences while retaining theoligomerization domain [Hallenberger et al. (1993), infra]. As can beappreciated by the skilled artisan, the term "oligomerization domain" isused functionally, to refer to a portion of gp41 that permitsoligomerization of env proteins, i.e., there is sufficient structure foroligomerization. The pVenv4 vector encodes a truncated gp160 (also:gp160t, gp140) that was discovered to form a tertiary structure that issimilar to that of the processed gp41/gp120 oligomer (dimer, trimer ortetramer) as is present at the cell surface of HIV infected cells. Thistertiary structure is maintained in both secreted and membraneassociated form [Hallenberger et al., (1993)]. This vector is preferablyused as a parent vector for the substitution of alternative isolated envsequences.

In this Example, the preparation of each VVenv construct involves theuse of a pVenv4 and a wildtype vaccinia virus NYCDH, and appropriatehost cells, as is described in detail below.

pVenv4. The pVenv4 vector was previously prepared by the insertion of anHIV-1-envelope coding sequence into the pSC11 vaccinia virusrecombination vector [Hallenberger, et al., Virology 193:510-514 (1993);Chakrabarti et al., Mol. Cell Biology 5:3403-3409 (1985)]. The HIV-1sequence was derived from a laboratory stock of live virus. The sequencewas named "BH10" [Ratner et al., Nature 313:277-284 (1985)]. With PCRtechniques unique envelope sequences from HIV-1 infected patients may beamplified and substituted into the BH10 env sequence to create newvectors. For example, the following primers might be used for PCR.

(A) Sense, Position 5785 (SEQ ID NO:1): AGCAGAAGACAGTGGCAATGAGAGTGA.

(B) Antisense, Position 7694 (SEQ ID NO:2):CCACTCCATCCAGGTCATGTTATTCCAAAT.

(C) KpnI-Sense, position 5903 (SEQ ID NO:3):GTGGGTCACAGTCTATTATGGGGTACCTGTGT.

(D) BsmI-Antisense, position 7659 (SEQ ID NO:4):CCAGAGATTTATTACTCCAACTAGCATTCCAAGG.

(E) (optional) DraIII-Sense, position 6153 (SEQ ID NO:5):CCATGTGTAAAATTAACCCCACTCTGTG.

(F) (optional) Bsu36I-Anti-sense, position 6917 (SEQ ID NO:6):TACAATTTCTGGGTCCCCTCCTGAGG.

These primers are written 5 to 3. Restriction sites are underlined(numbered positions are based on the BH10 sequence [Ratner et al.,Nature 313:277-284 (1985)].

PCR Strategy. In order to produce new HIV-1 env constructs, thepolymerase chain reaction (PCR) is used to amplify 1800 base pairs (bp)of envelope gene from forty different HIV-1 patient samples. The PCRprimers represent well-conserved HIV-1 sequences and thus successfullyamplified env genes from many diverse HIV-1 patient samples. Theamplified DNA encompasses the entire gp120 protein except forapproximately 10 highly conserved amino acids at the protein's aminoterminus. All. envelope variable regions (V1-V5) are included in the PCRproducts. In addition, amplified sequences encode approximately 100amino acids beyond the cleavage site for gp120/gp41.

The PCR primers carrying the restriction enzyme sites for KpnI and BsmI,which flank the BH10 envelope gene sequence in pVenv4, are incorporatedinto the amplified DNA products.

First Round PCR. In a 500 μl microcentrifuge tube, mix:

1 μl Primer A (SEQ ID NO: 1), 300 ng/μl;

1 μl primer B (SEQ ID NO: 2), 300 ng/μl;

2.5 μl 10 mM of each of 4 dNTPs;

1 μg DNA;

10 μl 10X PCR buffer; and

HPLC H20 to 99 μl

Vortex taq stock and dispense 1 μl to PCR reaction. Mix well. Overlaywith mineral oil.

Run on a thermal-cycler as follows:

Incubate 95° C., 3 minutes to melt DNA.

Run 40 cycles: 95° C., 1 minute; 45° C., 2 minutes; 72° C., 3.5 minutes.

Second Round PCR: Prepare PCR reaction as above, but with primers C andD (SEQ ID NOS:3 and 4, respectively) and without the DNA. Bring thefinal solution to 95 μl. Overlay with mineral oil. With a plugged tip,remove 5 μl from the first PCR reaction (from below the oil). Mix thesample into the second reaction, below oil layer and begin cycles asbefore. Thirty cycles is usually appropriate. It can be desirable tomonitor the product by removing 2 μl for gel analysis after each 10cycles until a clear band is identified of approximately 1800 bp.

By using well-known substitution cloning techniques, pVenv4 derivativesthat express an env sequence from one of the 40 patients, instead of theBH10 envelope sequence, were generated. Briefly, the pVenv4 plasmid andPCR products are next cut with KpnI and BsmI, and the cut pVenv4 was runon an agarose gel and the large fragment isolated. The small fragment(1800 bp fragment) of BH10 env was discarded. The cut PCR product wasalso isolated and ligated to the large pVenv4 fragment to create achimeric envelope sequence, now containing 1800 bp of the variant envfrom the patient DNA. Following ligation of the PCR product and thepVenv products, bacterial host cells are transformed with the ligationmixture via any of a number of methods well-known in the art, including,e.g., electroporation, and recombinant colonies are picked and examinedby sequencing.

Plasmid pVenv4 or recombinants made with pVenv4 facilitates theinsertion of genes into the vaccinia virus genome by homologousrecombination between the thymidine kinase (Tk) gene of the wildtypevirus and the Tk sequences within the plasmid. Insertion of pVenv4 DNAinto the viral Tk locus yields a vaccinia virus with the HIV-1 envelopegene expressed under the control of the P7.5K early/late promoter. Thevirus is attenuated in growth activity due to the disruption of the Tklocus. An additional element of pVenv4 is the lacZ gene that encodesβ-galactosidase activity, lacZ activity can be used to select vacciniavirus recombinants (see below).

The envelope gene expressed by pVenv4 is truncated to exclude thetransmembrane/C-terminal gp41 sequence. The vector is expressed as anoligomeric structure that is found within cells and in secreted form.

Vaccinia virus-NYCDH. Each new, substituted plasmid is individuallyrecombined with wildtype vaccinia virus NYCDH. This virus was obtainedfrom A.T.C.C. (Accession No. VR-325) and was plaque-purified prior touse (Buck, C., and Paulino, M. S., eds., American Type CultureCollection Catalogue of Animal Viruses and Antisera, Chlamydiae andRickettsiae, 6th Ed., American Type Culture Collection, Rockville, Md.(1990), p. 138).

Bacterial host cells. The plasmid may be grown on any suitable host, asknown in the art [see, e.g., Ausubel, infra (1995 rev), §§ 16.15-16.19].A non-limiting example is DH5α cells.

TK-deficient cells. The transformation and vaccinia virus substitutionis done on the human Tk⁻ 143B cell line, which is a TK-deficientderivative of the human cell line R970-5, an osteosarcoma cell line[Rhim et al. (1975), infra] that supports the growth of VV [Weir et al.(1982), infra]. Each vaccinia virus recombinant containing a unique HIVenv gene sequence is selected based on expression of the lacZ gene(Virus plaques are overlayed with Bluo-gal and selected forβ-galactosidase activity as judged by the development of a blue color).Two rounds of PCR can be performed.

Example 2

Preparation of Polyenv Vaccine

Vero Cells. The final manufacturing step is to grow n VVenv constructson Vero cells newly purchased from the A.T.C.C. (Accession No. CCL81 orX38) and cloned and expanded for virus growth. The Vero cell line hasbeen approved by the World Health Organization for vaccine development[Hay, R., et al., eds., American Type Culture Collection Catalogue ofCell Lines and Hybridomas, 7th Ed., American Type Culture Collection,Rockville, Md. (1992), page 48].

Vero cells are grown with Dulbecco's Modified Eagles Medium(Bio-Whittaker), a glutamine supplement (Bio-Whittaker) andheat-inactivated fetal calf serum (Hyclone, Inc.). Alternatively,serum-free media can be used. Each VVenv construct is inoculated onto aseparate confluent layer of Vero cells and harvested when cellsdemonstrate cytopathic effects due to virus infection. Cell extracts arewashed extensively with PBS (Bio-Whittaker) after harvest and beforefreezing. The cells are then broken open by freeze-thawing, sonicationor centrifuging at low speed in a centrifuge (optional). Aliquots ofsupernatant are then stored at -70° C. Envelope protein is present inthe lysate at sufficient concentrations to elicit HIV envelopeprotein-specific antibody (as detectable by ELISA) in mammal models,even if VV is attenuated, e.g., prep is heated to 60° C., 1 hr.

The Vaccine Product. Each virus (VVenv construct) stock from Vero cellsis individually frozen and subsequently titered and safety tested. Aftertests have been completed, aliquots of each virus are mixed to yield astock vaccine of 10⁸ total pfu/ml ("pfu" stands for plaque-formingunits). If 40 VVenv constructs are utilized, each VVenv is preferablyequally represented, each VVenv used at a titer of 2.5×10⁶ pfu/ml in thevaccine product. This should yield 1×10⁸ total pfu.

Evaluation of Polyenv Vaccine

Mice. Mice can be infected with an intraperitoneal injection of 1×10⁷pfu env-expressing VV. Antibody can be identified by HIV ELISA orneutralization assays, as described above, three weeks after VVinjections.

Prior to manufacture of the polyenv vaccine for human use, a similargroup of viruses has been prepared for the purpose of vaccine testing inmice. These viruses were administered to mice either by theintraperitoneal or subcutaneous route. We then tested serumHIV-1-specific antibody serum was tested for activity in anenzyme-linked immunosorbant assay (ELISA). The assay involved theplating of whole, disrupted HIV-1 (HTLV_(IIIB)) on ELISA plates and theblocking of plates with bovine serum albumin. Serum samples were thenadded at dilutions of 1:100, 1:1,000 and 1:10,000 in phosphate-bufferedsaline. The assay was developed with an alkaline-phosphatase-conjugatedgoat-anti-mouse immunoglobulin antibody and p-nitrophenyl phosphate. Thecolor reaction was stopped with a sodium hydroxide solution, and theoptical density reading was taken on an ELISA plate reader at 405 nm.

As shown in FIG. 2, a single inoculation with cell lysate preparation of10⁶ -10⁷ pfu vaccinia virus (containing a single HIV-1/envelope proteinencoding sequence and membrane bound expressed envelope protein)elicited a strong antibody response toward HIV-1 that was sustainedthroughout the experimental time course of six months. Such an antibodyresponse was significantly higher than previously reported with otherimmunizations. This high antibody response may be attributed to thepresence of membrane bound envelope protein in a vaccine preparation. Asshown in FIG. 3, these responses were dose dependent. Lower responseswere seen in mammals given a dose of 10⁶ pfu than in mammals given adose of 10⁷ pfu.

Mixtures of vaccinia viruses expressing different HIV-1 envelopeproteins were also prepared. When mice received 10⁷ pfu of a mixture offive viruses, their responses were essentially identical in magnitude toresponses generated against 10⁷ pfu of a single vaccinia virusrecombinant (FIG. 4). The mixing of numerous env-expressing vacciniaviruses in high numbers has not been reported, and is expected toprovide broad spectrum of neutralizing antibody.

Humans. Tests of the mixed virus stock are performed prior to clinicaltrials, the first of which will be for the purpose of dose escalationand safety testing.

The clinical trials will be a dose escalation study involving theassembly of four volunteer groups. Each group receives one of thefollowing vaccine doses:

(1) 2×10⁴ pfu

(2) 2×10⁵ pfu

(3) 2×10⁶ pfu

(4) 2×10⁷ pfu

Each volunteer receives the mixed virus vaccine in 0.5 ml saline,administered by a subcutaneous injection.

Example 3

Induction of Primary Isolate Neutralizing Immunity with aMulti-envelope, Vaccinia Virus-based Hiv-1 Vaccine in Chimpanzees

The population of HIV-1 isolates is armed with a sophisticated array ofenvelope proteins. Env proteins are the sole virally-encoded externalproteins and targets of neutralizing antibody activity, yet antibodieselicited toward one isolate will not necessarily neutralize another. Forthis reason, we have prepared an HIV-1 vaccine cocktail, PolyEnv,expressing numerous Env proteins. Vaccine production began with thepreparation of thirty distinct VV-recombinants, each expressing adistinct Env protein. VVenv were then tested, individually and incombination (PolyEnv) in a chimpanzee model. Four chimpanzees wereimmunized subcutaneously with three injections of single VVenv (Chimps 1and 2) or PolyEnv (Chimps 3 and 4) followed by one intramuscularinjection with recombinant gp120/gp41 protein in alum. Safety wasdemonstrated in all four animals, only two of which showed signs ofulceration at the injection site. Serum samples were monitored bynumerous tests for HIV-binding and neutralization. The antibodies ofchimps 3 and 4 demonstrated the highest quality of antibody activity.Neutralizing function was demonstrated both against a laboratory isolateand a primary isolate of HIV-1, neither of which were specificallyrepresented in the vaccine. Thus, the priming of lymphocytes with mixedenv proteins thus provides a promising method by which high-qualityantibodies may be elicited against diverse HIV-1.

Materials and Methods

pVenv4, a VV recombination vector. pVenv4 was previously prepared by theintroduction of a stop codon into the BH10-env sequence, and theinsertion of the modified BH10 envelope gene (env) into pSC11. pVenv4expressed an Env protein product that was truncated at amino acid 640,and was capable of both secretion and oligomerization. The production ofa recombinant VV, Vvenv4, expressing this truncated BH10 Env protein hasbeen described previously [Hallenberger et al., Virology 193:510-514(1993)].

PCR for the amplification of env sequences from HIV-1-infectedindividuals. PCR was used to amplify HIV env sequences. Generally,samples derived from the blood of HIV-1 infected individuals, taken atfirst diagnosis for HIV. Other samples were from individuals withclinical symptoms of AIDS, or from products provided by the AIDSresearch and reference reagent repository. For blood samples, DNA wasfirst prepared by the dropwise addition of blood or infected cells intoan SDS-based cell lysis buffer and incubation at 65 degrees C. for 30min. Pronase was added at a concentration of 0.5 mg/ml and the lysatewas further incubated at 45 degrees C. overnight. Two phenol extractionswere followed by ethanol precipitation, and resuspension of DNA inwater.

Two rounds of PCR were performed with all DNA samples by standardmethods. Primer sequences were chosen based on the published BH10sequence [Ratner et al., Nature 13:277-284 (1985)]. To obtain fragmentsincluding sequences from all variable regions and a portion of gp41, PCRprimers as described in Example 1 were used. PCR products weresubsequently cloned by substitution into the pVenv4 vector usingstandard methods. Sequencing was performed on the novel plasmids by useof the Sanger method and primer ccatgtgtaaaattaaccccactctgtg (SEQ IDNO:5).

Preparation of VVenv. Novel VV recombinants (VVenv) were prepared by thetransfection of VV (NYCDH, ATCC)-infected cells with the newlysubstituted recombination plasmids (see above). Transfectam (Promega)and Lipofectamine (Gibco, BRL) were used to facilitate transfection,following the manufacturer's recommendations. VV were then plaquepurified.

Immunizations. VVenv-infected cell lysates were administered tochimpanzees with subcutaneous injections. VVenv were either used singly,or in combination. The total quantities of VV by pfu were similar ineach injection (approximately 10⁷ pfu) per animal. Intramuscularinjections were with a mixture of approximately 40 micrograms gp120 (Cat# 12101, Intracel, Cambridge, Mass.), 20 micrograms of gp41 (Cat#036001, Intracel) and 500 micrograms alum (Rehsorptar Aluminumhydroxide Adsorptive Gel, Intergen Co., Purchase, N.Y.) per inoculum.

ELISAs. Five ELISAs were performed as follows: ELISA #1 The Abbottclinical ELISA was purchased from Abbott Laboratories and performed asrecommended by the manufacturers (HIVAB HIV-1/HIV-2 (rDNA) EIA, AbbottLaboratories, Abbott Park, Ill.). ELISA #2: ELISAs were performed byplating recombinant Mn-gp160 (Quality Biological, Inc. Gaithersburg,Md.) at one microgram/ml. Plates were blocked and tests were performedwith three-fold serial dilutions of sera. Plates were then washed andscored with alkaline phosphatase-conjugated anti-human IgG. ELISA 3:ELISA plates were coated with one microgram/ml of LAI-gp120 (CHO-derivedprotein, Intracel). Serum samples were plated after a 1:100 dilution andscored with alkaline phosphatase-conjugated anti-human IgG1 (Mouseanti-human IgG1-AP, cat #9050-04, Southern Biological Associates, Inc.,Birmingham, Ala.) and p-nitrophenyl phosphate. O.D. readings were takenat 405 nm. ELISA #4: The ELISA was performed as in assay #3, except thatplates were coated with one microgram/ml of IIIB-gp120(baculovirus-derived protein, Intracel, cat#12001, Cambridge, Mass.).ELISA#5: The ELISA was performed as in assay #3, except that plates werecoated with one microgram/ml of IIIB virus lysate (Organon Teknika Co.Durham, N.Y).

Neutralization assays. Neutralization assays were performed withlaboratory or primary isolates [Montefiori et al., J. Clin. Microbiol.26:231-237 (1988); Montefiori et al., Journal of Infectious diseases173:60-67 (1996)]. Laboratory isolates: Virus was mixed with a 1:20dilution of each serum sample, and plated on MT-2 or CEM-x174 cells.Neutral red stain was used to assess the viability of cells. A 35-40%reduction in cell death compared to control cultures was defined aspositive deflection. Primary Isolates: Virus was mixed with a 1:4dilution of each serum sample, and plated on PHA-stimulated PBMC. Assayswere scored for p24. A reduction of infectivity of at least 75% comparedto control cultures was required for a positive score.

Results

Preparation of novel VVenv recombinant vaccinia viruses. In order toprepare new VV recombinants (VVenv), each expressing a unique HIV-1 Envprotein, DNA was first isolated from HIV-1 samples. Most DNAs were fromthe blood of individuals who had shown no outward signs of disease andwere likely at first diagnosis for HIV-1 infection. Additional DNAs werefrom AIDS patients or from viruses provided by the AIDS Research andReference Reagent Repository. PCR was performed with primer pairsencompassing KpnI and BsmI restriction sites. Fragments were thensubstituted into the pVenv4 vector portrayed in FIG. 5 (pVenv4originally expressed a truncated HIV-1 protein, BH10) at KpnI and BsmIrestriction sites. In this way, gp120 (V1-V5) and gp41 sequences fromBH10 were replaced by respective sequences in PCR products. With eachsubstituted plasmid, a new VV recombinant virus (VVenv) was prepared.

This method provided a simple means by which a great diversity of Envsequences could be incorporated into unique VV recombinants (VVenv).Interestingly, the majority of sequences were productive, suggestingthat Env sequences in proviral genomes (from which most PCR productsderived) were rarely defective. Enormous diversity exists among theVVenv used in the vaccine mixture.

Immunization of chimpanzees with single or mixed VVenv. Four chimpanzeeswere used for the testing of single and mixed VV-recombinant vaccines.The schedule of immunizations are shown in Table 2. The first threeinjections contained VV while the last injection contained a combinationof gp120, gp41 and alum, given intramuscularly. Chimps 1 and 2 receivedonly one vaccinia virus and respective envelope protein, given in eachof the first three injections. Chimps 3 and 4 received a mixture of 10recombinant VV (and respective Env in the first injection), tenadditional Env in the second injection, and 10 additional, unique Env inthe last immunization, yielding a total of 30 distinct vectors prior tothe protein boost. All chimps received similar quantities of totalvaccinia virus in plaque forming units and similar quantities of totalrecombinant proteins.

                  TABLE 2                                                         ______________________________________                                        Immunization schedule for chimpanzees with mixed VV recombinants                       Date   Injection                                                     ______________________________________                                        Chimps 1 and 2:                                                                          1/9/96   VV (Env #1)                                                          4/16/96  VV (Env #1)                                                          6/11/96  VV (Env #1)                                                          7/30/96  recombinant gp120 and recombinant                                             gp41 + alum.                                              Chimps 3 and 4:                                                                          1/9/96   VV (Env #1-10)                                                       4/16/96  VV (Env #11-20)                                                      6/11/96  VV (Env #21-30)                                                      7/30/96  recombinant gp120 and recombinant                                             gp41 + alum.                                              ______________________________________                                    

Recombinant VVenv may be administered without lesion eruption. All fourchimps were monitored for signs of systemic disease and lesion formationat the site of infection. Animals were analyzed on a daily basis fordiarrhea, rhinorrhea, coughing, sneezing, rapid respiration, lethargy,restricted movement and loss of appetite. None of these signs wereevident in any animal at any time. Photographs taken of the injectionsites at regular intervals after the first VV immunization showedswelling evident in all four animals. Mild lesions appeared at theinjection sites on chimps 1 and 3 typifying a smallpox vaccination,while no lesions were evident on chimps 2 and 4. No disease symptoms,swelling or lesions were evident in the second, third or fourthinjections in any animal, demonstrating that VV-specific immunity hadbeen elicited by the first inoculation.

Injections of VVenv followed by protein booster immunizations yieldedELISA-positive antibody. HIV-specific antibodies were monitored duringthe immunization scheme by five different ELISAs. ELISAs were used tomeasure the relative quality, rather than absolute quantity ofantibodies in each animal. In most cases, tests were with virusfragments that lacked the three-dimensional and oligomeric structuretypical of native Env. In these cases, ELISAs would be expected to bindonly a subset of HIV-specific antibodies. Results obtained with theAbbott ELISA are shown in FIG. 6. Chimp 3 exceeded the cut-off forpositivity after the first VV immunization, while chimp 4 exceeded thecut-off after the second VVenv immunization. The responses of chimps 3and 4 at the end of the immunization scheme far exceeded those of chimps1 and 2. In ELISA #2 with MN gp160, chimp 3 was the only high responder.This response occurred prior to the protein boost, and was not perturbedby the booster injection. The response to CHO-LAI bound to an ELISA(ELISA #3) plate using the same antigen as that used for the purifiedprotein boost, showed only chimp 3 responded strongly. In ELISA #4 withIIIB-gp120 plate bound, Chimp 2 showed a high background and, perhapsdue to the high background, the highest response value of all animals.Responses to the fifth ELISA, Organon Technika IIIB virus lysate, werepositive with sera from all four animals.

Neutralization responses toward primary and laboratory isolates.Neutralization assays were performed with sera from each animal againstlaboratory and primary isolates. The first assay was performed on aT-cell line, while the latter assay was performed on sero-negativePHA-stimulated PBMC. In all cases, the isolates did not match thoserepresented in the HIV-1 vaccines.

As demonstrated in Table 3, samples from chimp 2, chimp 3 and chimp 4yielded a positive deflection (35-40% inhibition in virus growth)against the MN laboratory isolate in T cells. Assays with two otherlaboratory viruses (one IIIB [Lockey et al., Aids Res Hum Retroviruses12:1297-1299 (1996)] and one SF2 stock) did not score positively withany sample. The results of neutralization assays [Montefiori et al.,1988, supra; Montefiori et al., 1996, supra] with four primary isolatestested on PHA-stimulated PBMC are shown. Virus is considered difficultto neutralize in these assays, as patient sera often yield negativeresults, even when 1:2 dilutions are used [Fenyo et al., AIDS10:S97-S106 (1996); Moore and Ho, AIDS 9:S117-S136 (1995); Montefiori etal., 1996, supra]. Interestingly, a 1:4 dilution of chimp 4 serum wasable to neutralize one of the test primary isolates. The situationdiffered from the experiences of others with Env vaccines, as in mostprevious cases, sera from Env-immunized individuals have yieldednegative results in primary isolate neutralization assays [Steele,Journal of NIH research 6:40-42 (1994); Moore, Nature 376:115 (1995)].

                  TABLE 3                                                         ______________________________________                                        Neutralization by chimp antisera of viruses                                   not specifically represented in vaccine                                       Isolate  Chimp 1  Chimp 2    Chimp 3                                                                              Chimp 4                                   ______________________________________                                        Laboratory                                                                             --       Positive   Positive                                                                             Positive                                  strain MN         deflection deflection                                                                           deflection                                Primary #1                                                                             --       --         --     --                                        Primary #2                                                                             --       --         --     --                                        Primary #3                                                                             --       --         --     Positive                                  Primary #4                                                                             --       --         --     --                                        ______________________________________                                    

Mixed VVenv elicit a higher quality of HIV-1 specific antibodies thansingle VVenv. The results of ELISA and neutralization assays aresummarized in Table 4 listing those chimps whose sera yielded the higherresponses in the seven tests described above. As may be noted from thetable, chimps 3 and 4 scored positively in a composite of five out ofseven tests, while chimps 1 and 2 scored positively in only three out ofseven. This result may reflect a higher quality of antibodies elicitedby Poly Env as compared to single Env vaccines.

                  TABLE 4                                                         ______________________________________                                        Summary of ELISA and neutralization assays                                              Higher responses among                                                                        Higher responses among                              Assay     chimps given a single VV                                                                      chimps given mixed VV                               ______________________________________                                        Abbott (IIIB-             Chimp 3 and Chimp 4                                 gp41)-ELISA #1                                                                MNgp160BAC                Chimp 3                                             ELISA #2                                                                      IIIB-gp120-BAC-                                                                         Chimp 2                                                             ELISA #3                                                                      LaI-gp120-CHO-            Chimp 3                                             ELISA #4                                                                      III b Virus lysate                                                                      Chimp 1 and Chimp 2                                                                           Chimp 3 and Chimp 4                                 ELISA #5                                                                      Lab Isolate-                                                                            Chimp 2         Chimp 3 and 4                                       neutralization                                                                (deflection)                                                                  Primary Isolate-          Chimp 4                                             neutralization                                                                ______________________________________                                    

Discussion

Experiments described in this Example were designed to test the safetyof a vaccinia virus-based HIV-1 vaccine and to compare the efficacy ofpriming with envelope cocktails and single envelope vaccines. Resultsdemonstrated first, that vaccinia virus could be used as an immunogenwithout inducing an open lesion, and secondly, that a great breadth ofHIV-1-specific activity could be elicited with the envelope cocktail.

The chimpanzee model allowed us to examine the safety of PolyEnv inprimates. We were particularly interested to determine the extent ofopen lesion formation, as VV inoculations could pose a threat of livevirus transfer to unimmunized individuals. In the case of HIV, this is aserious concern in that an AIDS patient may not be capable of blockingthe VV infection. To address this concern, we tested the use ofsubcutaneous vaccinations in chimpanzees, questioning whether an openlesion could be avoided. Indeed, only two of the four chimpanzeesdemonstrated open lesions. Similar results were observed whensubcutaneous inoculations of the NYCDH vaccinia virus stock were used inclinical trials of the small pox vaccine [Connor et al., Journal ofInfectious diseases 135:167-175 (1977); Benenson et al., Journal ofInfectious diseases 135:135-144 (1977)].

It is likely that with additional attention to the injection procedureand follow-up care of the injection site, open lesions may be avoided inall cases. These results demonstrate that safety issues need notpreclude the use of vaccinia virus as an HIV-1 vaccine vector.

Envelope cocktails have been tested in mouse (Example 2) and rabbitexperiments. In the mouse experiments, anti-HIV antibodies weremonitored after a single injection of VVenv, while in rabbits, VVenvwere used to boost responses elicited with DNA-based. Experimentsindicated that HIV-l specific antibodies could be elicited or boostedwith VVenv, and that primary isolates could be neutralized by theantibody response. To examine the potential of mixed VVenv (PolyEnv),chimpanzees were divided into two groups. The first two chimps receivedonly one VVenv while chimps 3 and 4 received cocktails composed of atotal of thirty different VVenv.

After having received vaccinia virus immunizations, all four chimps weregiven a booster with a single gp120/gp41 protein mix in alum. The serafrom each of the four chimpanzees were tested in five different ELISAs,each utilizing a different fragment and/or configuration of Env.Interestingly, chimps 1 and 2 as a composite responded strongly in onlyone of these ELISAs, whereas the sera from chimps 3 and 4 as a compositeresponded strongly in 4 such assays. As each assay measured only afraction of the HIV-1 specific antibody in each animal, results likelyreflected the superior breadth of antibody binding activities elicitedby the mixed vaccine.

Neutralization assays were also performed both against laboratory andprimary isolates. Interestingly, a positive response against a primaryisolate was noted in chimp 4, even though the primary isolate had notbeen specifically represented in the vaccine mix. Again, these resultsdemonstrated a greater breadth of antibodies elicited by the PolyEnvvaccine cocktail. Increase in the antigen complexity of a vaccine mightbe expected to lead to an increased diversity of lymphocyte andrespective antibody responses.

The demonstration that neutralizing antibodies can be elicited against aprimary isolate that is not represented in the vaccine demonstrates thatlinearly distinct proteins share conformational structures. This notionis also demonstrated by the immune responses of HIV-1-infected patients,in that any two individuals who are exposed to a myriad of mutuallyexclusive viruses, are generally protected from superinfection whencross-exposure occurs. The use of PolyEnv represents a first attempt ina chimpanzee system to mimic the situation in HIV-1 patients. That is,neutralizing antibodies are elicited with a large array of, rather thana single, Env protein.

In summary, we have tested an VV-based HIV-1 vaccine cocktail calledPolyEnv in a chimpanzee model. This Example has demonstrated:

1) VV could be used as a vaccine without inducing an open skin lesion;

2) a great breadth of HIV-1 specific antibody activities could beelicited with this vaccine; and

3) a cocktail of Env constructs (PolyEnv) yielded a superior quality ofHIV-specific antibodies as compared to a single Env construct.

Vaccinia virus has long been known to be a potent vaccine, both inwildtype form and recombinant form. The strength of VV lies in its powerto recruit both the B- and cytotoxic T-lymphocyte compartments of theimmune response. VV has comprised the only vaccine capable oferadicating a disease (smallpox) from the human population. The data inthis Example indicate that recombinant VV vectors will contribute to thefuture control of HIV-1.

Example 4

Preparation of a Bi-Functional Plasmid

DNA vaccines have been shown to elicit strong antibody and CTL responsesin several, distinct systems (influenza, HIV-1, etc.). DNA-basedinfluenza and HIV-1 vaccines are already in clinical trials with healthyadult volunteers. Vaccinia virus also serves as a strong base forvaccination programs. In fact, vaccinia virus has been the only vaccineable to eradicate a disease (small pox) from the human population.Numerous recombinant vaccinia viruses have elicited protective immuneresponses as demonstrated in animal studies. The data shown abovedemonstrate the effectiveness of a polyenv vaccine, and of combiningvaccination strategies, e.g., DNA vaccines and viral vaccines.

A bi-functional plasmid that can act both as a DNA vaccine and a VVrecombinant vector is constructed. FIG. 7 shows a map of this plasmid,which includes a CMV promoter for expression in mammalian cells, andvaccinia early and late promoters for preparation of recombinantvaccinia. The direct injection of purified plasmid DNA would be used toelicit immune responses against an HIV env protein in test subjects. Theplasmid would also be used to prepare and test live, recombinantvaccinia viruses as HIV env protein immunization vehicles.

Subjects could potentially be vaccinated with a multi-tiered regimen,comprised both of DNA vaccination(s) and recombinant vaccinia virusimmunization(s), given in any order, in single or multiple injectionsand/or in conjunction with additional vaccine vehicles.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

It is further to be understood that all base sizes or amino acid sizes,and all molecular weight or molecular mass values, given for nucleicacids or polypeptides are approximate, and are provided for description.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

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    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 7                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 #             27   AATG AGAGTGA                                               - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 30 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 #           30     ATGT TATTCCAAAT                                            - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 32 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 #          32      TATG GGGTACCTGT GT                                         - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 34 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 #        34        CCAA CTAGCATTCC AAGG                                       - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 28 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 #             28   CCCC ACTCTGTG                                              - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 26 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 #              26  CCTC CTGAGG                                                - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 880 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: both                                                  -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - Lys Glu Gln Lys Thr Val Ala Met Arg Val Ly - #s Glu Ser Gln Met Lys         #                15                                                           - Lys Gln His Leu Trp Arg Trp Gly Trp Arg Tr - #p Gly Thr Met Leu Leu         #            30                                                               - Gly Leu Met Ile Cys Ser Ala Thr Glu Lys Le - #u Trp Val Thr Val Tyr         #        45                                                                   - Tyr Gly Val Pro Val Trp Lys Glu Ala Thr Th - #r Thr Leu Phe Cys Ala         #    60                                                                       - Ser Asp Ala Lys Ala Tyr Asp Thr Glu Val Hi - #s Asn Val Trp Ala Thr         #80                                                                           - His Ala Cys Val Pro Thr Asp Pro Asn Pro Gl - #n Glu Val Val Leu Val         #                95                                                           - Asn Val Thr Glu Asn Phe Asn Met Trp Lys As - #n Asp Met Val Glu Gln         #           110                                                               - Met His Glu Asp Ile Ile Ser Leu Trp Asp Gl - #n Ser Leu Lys Pro Cys         #       125                                                                   - Val Lys Leu Thr Pro Leu Cys Val Ser Leu Ly - #s Cys Thr Asp Leu Lys         #   140                                                                       - Asn Asp Thr Asn Thr Ser Asn Asn Val Thr Se - #r Ser Ser Trp Gly Arg         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Asn Ile Met Glu Glu Gly Glu Ile Lys Asn Cy - #s Ser Phe Asn Ile Ser         #               175                                                           - Thr Ser Ile Arg Gly Lys Val Gln Lys Glu Ty - #r Ala Phe Phe Tyr Lys         #           190                                                               - Leu Asp Ile Ile Pro Ile Asp Lys Gly Asn As - #p Ser Asn Asp Thr Thr         #       205                                                                   - Ser Tyr Lys Phe Thr Leu Thr Ser Cys Asn Th - #r Ser Val Ile Thr Gln         #   220                                                                       - Ala Cys Pro Lys Val Ser Phe Glu Pro Ile Pr - #o Ile His Tyr Cys Ala         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Pro Ala Gly Phe Ala Ile Leu Lys Cys Asn As - #n Lys Thr Phe Asn Gly         #               255                                                           - Thr Gly Pro Cys Thr Asn Val Ser Thr Val Gl - #n Cys Thr His Gly Ile         #           270                                                               - Arg Pro Val Val Ser Thr Gln Leu Leu Leu As - #n Gly Ser Leu Ala Glu         #       285                                                                   - Glu Glu Val Val Ile Arg Ser Ala Asn Phe Th - #r Asp Asn Ala Lys Thr         #   300                                                                       - Ile Ile Val Gln Leu Asn Gln Ser Val Glu Il - #e Asn Cys Thr Arg Pro         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Asn Asn Asn Thr Arg Lys Ser Ile Arg Ile Gl - #n Arg Gly Phe Gly Arg         #               335                                                           - Ala Phe Val Thr Ile Gly Lys Ile Leu Gly As - #n Met Arg Gln Ala His         #           350                                                               - Cys Asn Ile Ser Arg Ala Lys Trp Asn Asn Th - #r Leu Lys Gln Ile Asp         #       365                                                                   - Ser Lys Leu Arg Glu Gln Phe Gly Asn Asn Ly - #s Thr Ile Ile Phe Lys         #   380                                                                       - Gln Ser Ser Gly Gly Asp Pro Glu Ile Val Th - #r His Ser Phe Asn Cys         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Gly Gly Glu Phe Phe Tyr Cys Asn Ser Thr Gl - #n Leu Phe Asn Ser Thr         #               415                                                           - Trp Phe Asn Ser Thr Trp Ser Thr Lys Gly Se - #r Asn Asn Thr Glu Gly         #           430                                                               - Ser Asp Thr Ile Thr Leu Pro Cys Arg Ile Ly - #s Gln Ile Ile Asn Met         #       445                                                                   - Trp Gln Glu Val Gly Lys Ala Met Tyr Ala Pr - #o Pro Ile Ser Gly Gln         #   460                                                                       - Ile Arg Cys Ser Ser Asn Ile Thr Gly Leu Le - #u Leu Thr Arg Asp Gly         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Gly Ala Asn Glu Asn Asn Glu Ser Glu Ile Ph - #e Arg Pro Gly Gly Gly         #               495                                                           - Asp Met Arg Asp Asn Trp Arg Ser Glu Leu Ty - #r Lys Tyr Lys Val Val         #           510                                                               - Lys Ile Glu Pro Leu Gly Val Ala Pro Thr Ly - #s Ala Lys Arg Arg Val         #       525                                                                   - Val Gln Arg Glu Lys Arg Ala Val Gly Glu Il - #e Gly Ala Leu Phe Leu         #   540                                                                       - Gly Phe Leu Gly Ala Ala Gly Ser Thr Met Gl - #y Ala Ala Ser Met Thr         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Leu Thr Val Gln Ala Arg Gln Leu Leu Ser Gl - #y Ile Val Gln Gln Gln         #               575                                                           - Asn Asn Leu Leu Arg Ala Ile Glu Ala Gln Gl - #n His Leu Leu Gln Leu         #           590                                                               - Thr Val Trp Gly Ile Lys Gln Leu Gln Ala Ar - #g Ile Leu Ala Val Glu         #       605                                                                   - Arg Tyr Leu Lys Asp Gln Gln Leu Leu Gly Il - #e Trp Gly Cys Ser Gly         #   620                                                                       - Lys Leu Ile Cys Thr Thr Ala Val Pro Trp As - #n Ala Ser Trp Ser Asn         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Lys Ser Leu Glu Gln Ile Trp Asn Asn Met Th - #r Trp Met Glu Trp Asp         #               655                                                           - Arg Glu Ile Asn Asn Tyr Thr Ser Leu Ile Hi - #s Ser Leu Ile Glu Glu         #           670                                                               - Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln Gl - #u Leu Leu Glu Leu Asp         #       685                                                                   - Lys Trp Ala Ser Leu Trp Asn Trp Phe Asn Il - #e Thr Asn Trp Leu Trp         #   700                                                                       - Tyr Ile Lys Leu Phe Ile Met Ile Val Gly Gl - #y Leu Val Gly Leu Arg         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Ile Val Phe Ala Val Leu Ser Val Val Asn Ar - #g Val Arg Gln Gly Tyr         #               735                                                           - Ser Pro Leu Ser Phe Gln Thr His Leu Pro Il - #e Pro Arg Gly Pro Asp         #           750                                                               - Arg Pro Glu Gly Ile Glu Glu Glu Gly Gly Gl - #u Arg Asp Arg Asp Arg         #       765                                                                   - Ser Ile Arg Leu Val Asn Gly Ser Leu Ala Le - #u Ile Trp Asp Asp Leu         #   780                                                                       - Arg Ser Leu Cys Leu Phe Ser Tyr His Arg Le - #u Arg Asp Leu Leu Leu         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - Ile Val Thr Arg Ile Val Glu Leu Leu Gly Ar - #g Arg Gly Trp Glu Ala         #               815                                                           - Leu Lys Tyr Trp Trp Asn Leu Leu Gln Tyr Tr - #p Ser Gln Glu Leu Lys         #           830                                                               - Asn Ser Ala Val Ser Leu Leu Asn Ala Thr Al - #a Ile Ala Val Ala Glu         #       845                                                                   - Gly Thr Asp Arg Val Ile Glu Val Val Gln Gl - #y Ala Tyr Arg Ala Ile         #   860                                                                       - Arg His Ile Pro Arg Arg Ile Arg Gln Gly Le - #u Glu Arg Ile Leu Leu         865                 8 - #70                 8 - #75                 8 -       #80                                                                           __________________________________________________________________________

What is claimed is:
 1. A bi-functional plasmid that can serve as a DNAvaccine and a recombinant virus vector, comprising a heterologous geneand/or an insertion site for a heterologous gene under control of bothan animal expression control sequence, and a viral expression controlsequence.
 2. The bi-functional plasmid of claim 1 wherein the animalexpression control sequence is a cytomegalovirus immediate early (CMV)promoter.
 3. The bi-functional plasmid of claim 1 comprising aheterologous gene, wherein the heterologous gene is an env variant (EV)nucleic acid encoding both variable and constant regions of an envelopeprotein variant of an HIV envelope protein.
 4. The bi-functional plasmidof claim 2 comprising a heterologous gene, wherein the heterologous geneis an env variant (EV) nucleic acid encoding both variable and constantregions of an envelope protein variant of an HIV envelope protein. 5.The bi-functional plasmid of claim 1 wherein the virus expressioncontrol sequence is a vaccinia virus early promoter.
 6. Thebi-functional plasmid of claim 5 comprising a heterologous gene, whereinthe heterologous gene is an env variant (EV) nucleic acid encoding bothvariable and constant regions of an envelope protein variant of an HIVenvelope protein.
 7. The bi-functional plasmid of claim 1 wherein thevirus expression control sequence is a vaccinia virus late promoter. 8.The bi-functional plasmid of claim 7 comprising a heterologous gene,wherein the heterologous gene is an env variant (EV) nucleic acidencoding both variable and constant regions of an envelope proteinvariant of an HIV envelope protein.
 9. The bi-functional plasmid ofclaim 1 wherein the virus expression control sequence is both a vacciniavirus early promoter and a vaccinia virus late promoter.
 10. Thebi-functional plasmid of claim 9 comprising a heterologous gene, whereinthe heterologous gene is an env variant (EV) nucleic acid encoding bothvariable and constant regions of an envelope protein variant of an HIVenvelope protein.
 11. The bi-functional plasmid of claim 2 wherein thevirus expression control sequence is a vaccinia virus early promoter.12. The bi-functional plasmid of claim 11 comprising a heterologousgene, wherein the heterologous gene is an env variant (EV) nucleic acidencoding both variable and constant regions of an envelope proteinvariant of an HIV envelope protein.
 13. The bi-functional plasmid ofclaim 2 wherein the virus expression control sequence is a vacciniavirus late promoter.
 14. The bi-functional plasmid of claim 13comprising a heterologous gene, wherein the heterologous gene is an envvariant (EV) nucleic acid encoding both variable and constant regions ofan envelope protein variant of an HIV envelope protein.
 15. Thebi-functional plasmid of claim 2 wherein the virus expression controlsequence is both a vaccinia virus early promoter and a vaccinia viruslate promoter.
 16. The bi-functional plasmid of claim 15 comprising aheterologous gene, wherein the heterologous gene is an env variant (EV)nucleic acid encoding both variable and constant regions of an envelopeprotein variant of an HIV envelope protein.
 17. An immunogeniccomposition that can elicit an immune response to more than one but notnecessarily all of the env variants contained in the composition,comprising at least 4 different bi-functional plasmids, each encoding adifferent envelope protein variant (EPV) of a human immunodeficiencyvirus (HIV) envelope protein, whereina) the EPV contains both variableand constant regions; b) the immunogenic composition is capable ofeliciting at least one of a cellular and a humoral immune response in amammal against an HIV strain; and c) wherein the expression of each ofsaid different EPVs is under the control of both an animal expressioncontrol sequence, and a viral expression control sequence.
 18. Thebi-functional plasmid of claim 17 wherein the animal expression controlsequence is a cytomegalovirus immediate early (CMV) promoter.
 19. Thebi-functional plasmid of claim 18 wherein the virus expression controlsequence is a vaccinia virus early promoter.
 20. The bi-functionalplasmid of claim 18 wherein the virus expression control sequence is avaccinia virus late promoter.
 21. The bi-functional plasmid of claim 18wherein the virus expression control sequence is both a vaccinia virusearly promoter and a vaccinia virus late promoter.
 22. The bi-functionalplasmid of claim 17 wherein the virus expression control sequence is avaccinia virus early promoter.
 23. The bi-functional plasmid of claim 17wherein the virus expression control sequence is a vaccinia virus latepromoter.
 24. The bi-functional plasmid of claim 17 wherein the virusexpression control sequence is both a vaccinia virus early promoter anda vaccinia virus late promoter.